Detection of Enteroviral Infection in Paraffin-Embedded Tissue by the RNA Polymerase Chain Reaction Technique
Am J Clin Pathol 096: 568-571 (5)
NOV
Enterovirus; Polymerase Chain Reaction; Viral Diagnosis; Myocarditis; AMPLIFICATION; DNA
RW Redline/Case Western Reserve Univ/Inst Pathol/2085 Adelbert Rd/Cleveland, OH 44118| --- A method using the RNA polymerase chain reaction technique to diagnose infection retrospectively using single-stranded RNA enteroviruses in paraffin-embedded tissue blocks is reported. This method takes advantage of extreme sequence conservation in the 5' untranslated region of the enteroviral genome and uses two rounds of amplification with nested oligonucleotide primers, thus allowing rapid diagnosis without the use of radioactive reagents. The technique should prove useful in cases in which viral infection is suspected after histopathologic evaluation, when fresh or frozen tissues often are unavailable. The sensitivity of the method is demonstrated by successful amplification of Enterovirus 11 RNA extracted from 4-year-old liver tissue obtained at autopsy examination that was initially fixed and embedded 45 hours after death.
Detection of Immunoglobulin Gene Rearrangement in Acute and Chronic Lymphoid Leukemias of B-Cell Lineage by Polymerase Chain Reaction Gene Amplification
R Liang/Univ Hong Kong/Queen Mary Hosp/Dept Med/Hong Kong, Hong Kong| --- Polymerase chain reaction (PCR) was used to amplify the DNA fragments of the complementarity determining region 3 of the immunoglobulin (Ig) gene heavy chain from the leukemic cell specimens of patients with acute and chronic lymphoid leukemias of B-cell lineage. Two different pairs of primers were tested. Fourteen of the 17 (82%) cases of acute lymphoblastic leukemia (ALL), and all 15 cases (100%) of B-cell chronic lymphocytic leukemia, who had rearrangement of the Ig gene heavy chain by Southern analysis, were positive by PCR with either one or both pairs of primers. This technique was able to detect leukemic cells at the level of 0.1%. Applying it to study the remission marrow specimens following induction chemotherapy was more useful than morphology alone in predicting early relapse of the leukemia.
Marshall, T.; Williams, K.M.
1991
Biochemi
Artic/En
L34/46
Drug Interference in the Bradford and 2,2'-Bicinchoninic Acid Protein Assays
Anal Biochem 198: 352-354 (2)
NOV 1
BICINCHONINIC ACID; DYE-BINDING
T Marshall/Sunderland Polytech/Sch Pharmaceut & Chem Sci/Biochem Res Lab/Galen Bldg/Green Terrace/Sunderland SR2 7EE/Durham, England|
Ghatge, M.; Mawal, Y.; Gaikwad, S.; Deshpande, V.
1991
Biochemi
Artic/En
L34/46
Immunoaffinity Purification of Glucose Xylose Isomerase from Streptomyces
V Deshpande/Natl Chem Lab/Div Biochem Sci/Poona 411008/Maharashtra, India| --- A procedure was developed to purify glucose/xylose isomerase from cell extract of Streptomyces sp. NCIM 2730 using immunoaffinity chromatography. High-titer polyclonal antibodies were raised in rabbit using electrophoretically homogeneous glucose/xylose isomerase as an antigen. The specificity of antibodies was confirmed by double immunodiffusion, rocket electrophoresis, and Western-blot ELISA, which revealed the presence of a single immunoreactive protein with an M(r) of 40, 000. The antibodies recognized 2-3 antigenic determinants/mol of enzyme and were found to partially neutralize the enzymatic activity in an immunotitration experiment. The affinity gel was prepared by coupling antibodies at pH 10.0 to divinyl sulfone-activated Sepharose CL-4B. The glucose/xylose isomerase purified by immunoaffinity chromatography yielded 75% recovery with a single enzymatically active protein band on gel electrophoresis and showed specific activity of 16 U/mg. The crossreaction of the antibodies with glucose isomerase from other actinomycetes indicated that they share common epitopes.
Post-Embedding Immuno-Electron Microscopy with Rapid Freezing and Freeze-Substitution Techniques for the Localization of Manganese Superoxide Dismutase
Arch Histol Cytol 054: 465-469 (4)
OCT
TUMOR NECROSIS FACTOR; MN-SOD; CELLS; RAT
H Tatsumi/Sapporo Med Coll/Dept Anat/S1/W17/Chou Ku/Sapporo/Hokkaido 060, Japan| --- Dehydration of specimens with ethanol or acetone makes it impossible to detect manganese superoxide dismutase (Mn-SOD) by immunohistochemistry. To circumvent obstacles and demonstrate localization by post-embedding immuno-electron microscopy, a rapid freezing and freeze-substitution technique was employed using Lowicryl K4M embedding medium. This was effective enough to allow the specific observation of immunogold particles for Mn-SOD on the mitochondria of cardiac muscle cells and WI-38 cells (human normal fetal lung diploid cells). This method preserved the antigen -antibody binding activity of Mn-SOD even after dehydration. Therefore, rapid freezing and freeze-substitution is useful for post-embedding immuno-electron microscopy of Mn-SOD and can further be employed for other antigens previously difficult to detect by conventional methods.
Rubinsky, B.; Arav, A.; Fletcher, G.L.
1991
Biochemi
Artic/En
L34/48
Hypothermic Protection - A Fundamental Property of Antifreeze Proteins
J Lippincottschwartz/Nichhd/Cell Biol & Metab Branch/Bethesda, MD 20892| --- Addition of brefeldin A (BFA) to most cells results in both the formation of extensive, uncoated membrane tubules through which Golgi components redistribute into the ER and the failure to transport molecules out of this mixed ER/Golgi system. In this study we provide evidence that suggests BFA's effects are not limited to the Golgi apparatus but are reiterated throughout the central vacuolar system. Addition of BFA to cells resulted in the tubulation of the endosomal system, the trans-Golgi network (TGN), and lysosomes. Tubule formation of these organelles was specific to BFA, shared near identical pharmacologic characteristics as Golgi tubules and resulted in targeted membrane fusion. Analogous to the mixing of the Golgi with the ER during BFA treatment, the TGN mixed with the recycling endosomal system. This mixed system remained functional with normal cycling between plasma membrane and endosomes, but traffic between endosomes and lysosomes was impaired.
Hunziker, W.; Whitney, J.A.; Mellman, I.
1991
MicrCell
Artic/En
L34/48
Selective Inhibition of Transcytosis by Brefeldin-A in MDCK Cells
W Hunziker/Yale Univ/Sch Med/Dept Cell Biol/New Haven, CT 06510| --- Treatment of most cells with brefeldin A (BFA) leads to the retrieval of the Golgi complex to the endoplasmic reticulum, presumably reflecting an inhibition of cytoplasmic coat protein binding to Golgi membranes. Although BFA has been thought to act only on biosynthetic organelles, we now show that this drug also reversibly blocks polymeric immunoglobulin receptor-mediated transcytosis in MDCK cells. The action of BFA on transcytosis was selective, since internalization, recycling, and intracellular degradation were unaffected. The block occurred early on the transcytotic pathway, probably before the translocation of IgA-containing vesicles from the basal to the apical cytoplasm. Although BFA caused MDCK cell endosomes to become more tubular, the organization of the Golgi and binding of the 110 kd Golgi coat protein beta-COP was surprisingly unaffected. These results suggest that in MDCK cells, endocytic organelles contain a BFA-sensitive coat that regulates their organization and function even though the Golgi coat is BFA resistant.
Tian, Q.S.; Streuli, M.; Saito, H.; Schlossman, S.F.; Anderson, P.
1991
MicrCell
Artic/En
L34/48
A Polyadenylate Binding Protein Localized to the Granules of Cytolytic Lymphocytes Induces DNA Fragmentation in Target Cells
QS Tian/Harvard Univ/Sch Med/Dana Farber Canc Inst/Div Tumor Immunol/Boston, MA 02115| --- Cytolytic lymphocytes (CTLs) are characterized by their inclusion of cytoplasmic granules containing effector molecules such as perforin and the serine proteases. Here we describe the cDNA cloning and functional characterization of two related isoforms of a cytolytic granule protein designated TIA-1. Sequence analysis reveals that the 40 kd TIA-1 isoform (rp40-TIA-1) is structurally related to the poly(A)-binding proteins, possessing three RNA-binding domains and a carboxy-terminal, glutamine-rich auxiliary domain. The 15 kd TIA-1 isoform, the major species present in cytolytic granules, appears to be derived from the carboxy-terminal auxiliary domain of rp40-TIA-1 by proteolytic processing. Both natural and recombinant TIA-1 were found to induce DNA fragmentation in digitonin permeabilized thymocytes, suggesting that these molecules may be the granule components responsible for inducing apoptosis in CTL targets.
Mccown, B.H.; Joyce, P.J.
1991
*CurBook
Artic/En
L34/47
Automated Propagation of Microtubers of Potato --- In: Cell Culture and Somatic Cell Genetics of Plants, Vol 8 (Series: Cell Culture and Somatic Cell Genetics of Plants 8 (1991)), pp. 95-109. Editor(s): IK Vasil. Academic Press Inc, 1250 6TH Ave, San Diego, CA 92101. ISBN 0-12-715008-0
Cell Culture and Somatic Cell 008: 95-109 ()
BH Mccown/Univ Wisconsin/Dept Hort/Madison, WI 53706|
Luchtel, D.L.; Deyrupolsen, I.; Martin, A.W.
1991
MicrCell
Artic/En
L34/46
Ultrastructure and Lysis of Mucin-Containing Granules in Epidermal Secretions of the Terrestrial Slug Ariolimax-Columbianus (Mollusca, Gastropoda, Pulmonata)
DL Luchtel/Univ Washington/Dept Environm Hlth/Seattle, WA 98195| --- The giant mucous cells in the skin of the terrestrial banana slug Ariolimax columbianus secret intact granules containing mucins. Electron microscopy, after ultrarapid freezing and freeze-substitution in osmium, shows that the secreted granules are bounded by two distinct membranes, presumably derived from the Golgi apparatus and the plasmalemma. Relatively stable, intact granules can be obtained in great quantity in our in vitro system. Rapid lysis of the granules was induced by adenosine triphosphate. At much higher concentrations, adenosine diphosphate and 5'-adenylimido-diphosphate also caused lysis. Other nucleotides and related compounds, as well as 1,4,5-inositol triphosphate and molluscan neurotransmitters and neuropeptides, had no effect on the granules.The stability of secreted granules varied with the ionic composition of the isosmotic medium in which they were suspended. When the predominant cation in the medium was potassium, and calcium was also present, granules lysed if exposed to shear stress (stirring of the suspension). This did not occur if sodium was the major cation present. None of the other ions in the suspension media had detectable effects on the stability of the granules.
Ribeiro, J.M.C.; Endris, T.M.; Endris, R.
1991
Physiolo
Artic/En
L34/47
Saliva of the Soft Tick, Ornithodoros moubata, Contains Anti-Platelet and Apyrase Activities
Comp Biochem Physiol [A] 100: 109-112 (1)
RHODNIUS-PROLIXUS; IXODES-DAMMINI; AGGREGATION
JMC Ribeiro/Univ Arizona/Dept Entomol/Forbes Bldg 410/Tucson, AZ 85721| --- 1. Pilocarpine-induced saliva of the soft tick Ornithodoros moubata inhibits platelet aggregation induced by ADP or collagen, even when diluted 2000 times into platelet rich plasma. 2. Saliva contains apyrase (ATP-diphosphohydrolase) activity, which has an optimal pH of 7.0 for ADP and of 8.0 for ATP hydrolysis, respectively. Both Ca2+ and Mg2+ activate the reactions. 3. The mean specific activities for ATP and ADP hydrolysis at pH 7.5 were 0.97 and 0.74-mu-moles orthophosphate/min/mg protein. 4. These results, which demonstrate for the first time such activities in the saliva of soft ticks, support the hypothesis that the saliva of blood sucking arthropods serves an anti-hemostatic role during feeding and that large amounts of salivary apyrase activity have evolved independently in hematophagous arthropods.
Miller, W.; Barr, J.; Rudd, K.E.
1991
ExpBioMe
Artic/En
L34/46
Improved Algorithms for Searching Restriction Maps
W Miller/Penn State Univ/Dept Comp Sci/University Pk, PA 16802| --- We present algorithms for searching a DNA restriction enzyme map for a region that best matches a shorter 'probe' map. Our algorithms utilize a new model of map alignments, and extensive experiments prove our model superior to earlier approaches for certain applications. Let M be the number of map sites and P be the number of probe sites. Our first algorithm, which optimizes only over a restricted class of alignments, requires O(MP log P) worst-case time and O(M + P) space. Our second algorithm, which optimizes over all alignments, runs in O(MP3) time and O(M + P2) space, under reasonable assumptions about the distribution of restriction enzyme cleavage sites. Combining the algorithms gives a map-searching method that optimizes over all alignments in O(MP log P) time in practice. The algorithms' effectiveness is illustrated by searches involving a genomic restriction map of Escherichia coli.
Berger, M.P.; Munson, P.J.
1991
ExpBioMe
Artic/En
L34/46
A Novel Randomized Iterative Strategy for Aligning Multiple Protein Sequences
Comput Appl Biosci 007: 479-484 (4)
OCT
ALIGNMENT; ALGORITHM; TREES
PJ Munson/NIH/Div Comp Res & Technol/Analyt Biostat Sect/Bldg 12A/Room 2009/Bethesda, MD 20892| --- The rigorous alignment of multiple protein sequences becomes impractical even with a modest number of sequences, since computer memory and time requirements increase as the product of the lengths of the sequences. We have devised a strategy to approach such an optimal alignment, which modifies the intensive computer storage and time requirements of dynamic programming. Our algorithm randomly divides a group of unaligned sequences into two subgroups, between which an optimal alignment is then obtained by a Needleman-Wunsch style of algorithm. Our algorithm uses a matrix with dimensions corresponding to the lengths of the two aligned sequence subgroups. The pairwise alignment process is repeated using different random divisions of the whole group into two subgroups.Compared with the rigorous approach of solving the n-dimensional lattice by dynamic programming, our iterative algorithm results in alignments that match or are close to the optimal solution, on a limited set of test problems. We have implemented this algorithm in a computer program that runs on the IBM PC class of machines, together with a user-friendly environment for interactively selecting sequences or groups of sequences to be aligned either simultaneously or progressively.
Taylor, P.; Rosenberg, P.; Samsonova, M.G.
1991
ExpBioMe
Artic/En
L34/46
A New Method for Finding Long Consensus Patterns in Nucleic Acid Sequences
P Taylor/Inst Virol/MRC Virol Unit/Glasgow G11 5JR, Scotland| --- We describe a fast computer algorithm for identifying consensus patterns in DNA sequences. The method requires no prior assumptions about the consensus pattern other than its length. In particular no previous knowledge of the frequency or spacing of consensus patterns is required. However, a priori information about the shape of the consensus pattern, or invariability of individual positions, or the overall conservation level, can be utilized to enhance the selectivity and sensitivity of search. As the number of all possible consensus words increases very rapidly with length, comprehensive searches have usually been restricted to a maximum of 10-12 nucleotides, even when large mainframes are used. Our algorithm enables searching for consensus patterns of this order on current mid-range and powerful microcomputers. Searches may be conducted on single, long sequences or a set of possibly aligned shorter sequences. We give examples of identified consensus patterns in both prokaryotic and eukaryotic DNA sequences, along with some typical program timings.
Danckaert, A.; Chappey, C.; Hazout, S.
1991
ExpBioMe
Artic/En
L34/46
Size Leap Algorithm - An Efficient Extraction of the Longest Common Motifs from a Molecular Sequence Set - Application to the DNA Sequence Reconstruction
Comput Appl Biosci 007: 509-513 (4)
OCT
GEL READING DATA; COMPUTER-PROGRAM; PROTEINS; MULTAN
S Hazout/Univ Paris 07/Inserm/U263/Unite Rech Biomath & Biostat/2 Pl Jussieu/F-75251 Paris 05, France| --- We propose a new method, called 'size leap' algorithm, of search for motifs of maximum size and common to two fragments at least. It allows the creation of a reduced database of motifs from a set of sequences whose size obeys the series of Fibonacci numbers. The convenience lies in the efficiency of the motif extraction. It can be applied in the establishment of overlap regions for DNA sequence reconstruction and multiple alignment of biological sequences. The method of complete DNA sequence reconstruction by extraction of the longest motifs ('anchor motifs') is presented as an application of the size leap algorithm. The details of a reconstruction from three sequenced fragments are given as an example.
Lucas, K.; Busch, M.; Mossinger, S.; Thompson, J.A.
1991
ExpBioMe
Note/En
L34/46
An Improved Microcomputer Program for Finding Gene Family or Gene Family-Specific Oligonucleotides Suitable as Primers for Polymerase Chain Reactions or as Probes
JA Thompson/Max Planck Inst Immunbiol/Stubeweg 51/D-7800 Freiburg, Fed Rep Ger| --- We present here an easy-to-use computer program which finds oligonucleotides suitable as primers in polymerase chain reactions (PCR) or as probes for hybridization. In contrast to other programs used for this purpose, the additional advantage of this one is the possibility of directly detecting gene- as well as gene family-specific oligonucleotides. For this purpose, up to 200 different DNA sequences, of maximally 65 000 nucleotides each, can be scanned in a single search to ensure either single or multiple gene binding of the PCR primers or probes. Specific oligonucleotides for genes carrying internal repetitions and for single genes belonging to a set of highly conserved genes can also be detected. Many parameters such as exclusion of simple sequences, which are known to be highly repeated throughout various genomes or regions of stable secondary structures in both primer-primer and primer-template, can be taken into consideration and avoided. Furthermore, the G + C content and the length of the oligonucleotides can be changed in a broad range by the user.
Sibbald, P.R.; Sommerfeldt, H.; Argos, P.
1991
ExpBioMe
Note/En
L34/46
Automated Protein Sequence Pattern Handling and PROSITE Searching
Comput Appl Biosci 007: 535-536 (4)
OCT
PR Sibbald/European Molec Biol Lab/Postfach 102209/Meyerhofstr 1/D-6900 Heidelberg, Fed Rep Ger| --- The protein sequence searching program Scrutineer has been modified to search for targets from a file. We are distributing a reformatted file of PROSITES which can be read by Scrutineer. In addition, Scrutineer still accepts targets typed in interactively but can now write them out in the format required as input. Since the input format is the same as the output format, target management and re-use is simple.
IA Hope/Univ Leeds/Dept Pure & Appl Biol/Leeds LS2 9JT/W Yorkshire, England| --- A screen of gene expression patterns has been developed for the nematode Caenorhabditis elegans. Promoter-reporter gene fusions were constructed in vitro by ligating C. elegans genomic DNA fragments upstream of a lacZ gene. Patterns of beta-galactosidase expression were examined by histochemical staining of C.elegans lines transformed with the constructs. Beta-galactosidase expression depended on translational fusion, so constructs were assayed in large pools to expedite detection of the low proportion that were active. Expression in a variety of cell types and temporal patterns was observed with different construct pools. The most striking expression patterns were obtained when the beta-galactosidase activity was localized to subcellular structures by the C. elegans portion of the fusion protein. The active constructs of three selected pools were identified subsequently by an efficient combinatorial procedure. The genomic locations of the DNA fragments from the active constructs were determined and appear to define previously uncharacterized genetic loci.
A Fire/Univ British Columbia/Dept Zool/Vancouver V6T 2A9/BC, Canada| --- We have used an antisense strategy to effectively disrupt the expression of two genes encoding myofilament proteins present in C. elegans body wall muscles. DNA segments from the unc-22 and unc-54 genes have been placed in reverse orientation in vectors designed to produce RNA in body wall muscles. When the resulting plasmids are injected into oocytes, progeny with defects in muscle function are produced. These animals have phenotypes consistent with reduction and/or elimination of function of the gene to which antisense RNA has been produced: twitching and disorganization of muscle filaments for the unc-22 antisense constructs and lack of muscle tone, slow movement, and egg laying defects for the unc-54 antisense constructs. A fraction of the affected animals transmit the defective-muscle trait to subsequent generations. In these cases the transforming DNA is present at high copy number and cosegregates with the observed muscle defects. We have examined several of the unc-22 antisense plasmid transformed lines to determine the mechanistic basis for the observed phenotypes. The RNA product of the endogenous unc-22 locus is present at normal levels and this RNA is properly spliced in the region homologous to the antisense RNA. No evidence for modification of this RNA by deamination of adenosine to inosine was found. In affected animals the level of protein product from the endogenous unc-22 locus is greatly reduced. Antisense RNA produced from the transforming DNA was detected and was much more abundant than 'sense' RNA from the endogenous locus. These data suggest that the observed phenotypes result from interference with a late step in gene expression, such as transport into the cytoplasm or translation.
Pattern Formation in the Nematode Epidermis - Determination of the Arrangement of Peripheral Sense Organs in the C-Elegans Male Tail
Development 113: 515-526 (2)
OCT
Morphogenesis; Pattern Formation; Peripheral Sense Organs; POST-EMBRYONIC DEVELOPMENT; CAENORHABDITIS-ELEGANS; CELL; EXPRESSION; LINEAGE; PROTEIN; REGION
SW Emmons/Yeshiva Univ Albert Einstein Coll Med/Dept Molec Genet/1300 Morris Pk Ave/Bronx, NY 10461| --- The developmental process that determines the arrangement of ray sensilla in the Caenorhabditis elegans male tail has been studied. It is shown that the adult arrangement of rays is determined by the placement of ray cells at specific sites in the epidermis of the last larval (L4) stage. Placement of ray cells at specific epidermal sites results from the generation of neurons and support cells in the epidermis near to their final positions, and the subsequent refinement of these positions by an active mechanism involving specific cellular associations. Positions of ray cells and adjacent epidermal cells have been studied during ray development by means of indirect immunofluorescence staining with an antibody to a cell junctional antigen. Mutations are described in six genes that alter the adult arrangement of the rays, frequently resulting in fusion of rays. Changes in the adult pattern of rays in mutants appear to result from prior changes in the epidermal positions of ray cells, and for two mutants it is suggested that this may be due to the inappropriate clustering of processes from neurons and support cells of adjacent rays. Development of the wild-type arrangement of rays appears to require the specification of molecular differences between the rays that affect the specificity of their cellular associations.
Ofulue, E.N.; Candido, E.P.M.
1991
MolBiGen
Artic/En
L34/46
Molecular Cloning and Characterization of the Caenorhabditis-Elegans Elongation Factor-2 Gene (eft-2)
EPM Candido/Univ British Columbia/Fac Med/Dept Biochem/Vancouver V6T 1Z3/BC, Canada| --- A Caenorhabditis elegans lambda-ZAP cDNA library was screened using a fragment amplified from highly conserved regions of the mammalian and Drosophila elongation factor 2 (EF-2). Two types of cDNA clones were obtained, corresponding to two mRNA species with 3'-untranslated regions of 60 and 115 nucleotides, both encoding identical polypeptides. Sequence analysis of these clones and comparisons with hamster and Drosophila EF-2 sequences suggests that they encode C. elegans EF-2. Clone pCef6A, encoding the entire C. elegans EF-2 mRNA sequence including 45 nucleotides of 5'-untranslated region, contains a 2,556-bp open reading frame which predicts a polypeptide of 852 amino acid residues (M(r) 94,564). The deduced amino acid sequence is greater than 80% identical to that of mammalian and Drosophila EF-2. Conserved sequence segments shared among a variety of GTP-binding proteins are found in the amino-terminal region. The carboxy-terminal half contains segments unique to EF-2 and its prokaryotic homolog, EF-G, as well as the histidyl residue which is ADP-ribosylated by diphtheria toxin. The C. elegans protein contains a 12-amino-acid insertion between positions 90 and 100, and a 13-amino-acid deletion between positions 237 and 260, relative to hamster EF-2. Partial sequencing of a genomic clone encoding the entire C. elegans EF-2 gene (named eft-2) has so far revealed two introns of 48 and 44 bp following codons Gln-191 and Gln-250, respectively. Southern and Northern blot analyses indicate that eft-2 is a single-copy gene and encodes a 3-kb mRNA species which is present throughout nematode development.
A Draeger/Austrian Acad Sci/Inst Molec Biol/Billrothstr 11/A-5020 Salzburg, Austria| --- Two-dimensional gel analysis of basic proteins in developing human smooth muscle identifies calponin as a prominent marker of the differentiated phenotype. Adult tissue (human and mouse) typically expresses up to four calponin isoforms, three of which appear sequentially during fetal development: adult myometrial cells express the same three isoforms in primary culture in vitro and these are down-regulated, in reverse order, during the subsequent modulation of phenotype. Monospecific, polyclonal antibodies against calponin identify a lower molecular weight variant of calponin (L-calponin) that is strongly and specifically expressed in adult smooth muscles of the human urogenital tract. L-Calponin is down-regulated in benign smooth muscle derived tumors (leiomyoma) and is not expressed in primary cultures of normal uterine tissue.
AI Gragerov/Acad Sci USSR/Inst Molec Genet/Moscow 123182, USSR| --- Mutations in the rpoH gene, encoding sigma-32, an alternative factor required for transcription of the heat shock genes, result in the extensive aggregation of virtually all cellular proteins and formation of inclusion bodies both under stress and non-stress conditions. Inhibitors of protein synthesis suppress this aggregation, suggesting that newly synthesized proteins preferentially aggregate in rpoH mutants. These data suggest that the heat shock proteins are involved in acquisition of the soluble state (i.e. correct conformation) of the bulk of intracellular proteins after their translation.
Legagneux, V.; Morange, M.; Bensaude, O.
1991
Biochemi
Artic/En
L34/48
Heat Shock Increases Turnover of 90 kDa Heat Shock Protein Phosphate Groups in HeLa Cells
FEBS Lett 291: 359-362 (2)
OCT 21
Heat Shock; Heat Shock Protein; Protein Phosphorylation; HeLa Cell; GLUCOCORTICOID RECEPTOR; BINDING-PROTEINS; PHOSPHORYLATION; LIGAND
O Bensaude/Ecole Norm Super/Dept Biol/Biol Molec Stress Lab/46 Rue Dulm/F-75230 Paris 05, France| --- The 90 kDa heat shock protein (hsp90) is a major phosphoprotein which associates various other cellular polypeptides such as actin, calmodulin, steroid hormone receptors and certain protein-kinases. Little is known about the function of hsp90 in recovery from stress. In this report, we describe a dramatic increase in the rate of both phosphate uptake and dephosphorylation of hsp90 in HeLa cells submitted to acute stresses. This increased turnover of hsp90 phosphate groups might reflect a greater protein binding activity of hsp90 in stressed cells.
Jessopmurray, H.; Martin, L.D.; Gilley, D.; Preer, J.R.; Polisky, B.
1991
MolBiGen
Artic/En
L34/47
Permanent Rescue of a Non-Mendelian Mutation of Paramecium by Microinjection of Specific DNA Sequences
H Jessopmurray/Indiana Univ/Dept Biol/Program Molec Cellular & Dev Biol/Bloomington, IN 47405| --- The mutant Paramecium tetraurelia cell line d48 is unable to express the serotype A protein on its surface. Although the A gene is intact in the micronuclei of d48, the A gene copies in the macronucleus contain a large deletion eliminating virtually the entire coding sequence. Previous studies showed that microinjection of a plasmid containing the entire A gene into the macronucleus of d48 permanently restored A expression after autogamy. Together with other data, this result suggests that in wild type cells the A gene in the old macronucleus ensures the presence of a cytoplasmic factor that prevents A gene deletions at autogamy. In d48, where there are few, if any copies of the intact A gene in the old macronucleus, deletions occur during macronuclear formation. To elucidate the specific molecular mechanisms involved in this unusual phenomenon, we attempted to define the region(s) of the A gene necessary for rescuing d48. We show that microinjection of a 4.5-kb internal A gene fragment is sufficient for proper processing at autogamy and leads to permanent rescue of d48; i.e., the rescued strain is indistinguishable from wild type. Thus, rescue of d48 does not require upstream transcriptional control sequences, intact A mRNA or A serotype protein. We also show that various fragments of the A gene have the ability to rescue d48 to different extents, some being more efficient than others. We find no evidence to suggest that the A gene gives rise to a small stable RNA that might act as or encode a cytoplasmic factor. Molecular mechanisms that may be involved in the rescue of d48 are discussed.
Johnsen, R.C.; Baillie, D.L.
1991
MolBiGen
Artic/En
L34/47
Genetic Analysis of a Major Segment <LGV(Left)> of the Genome of Caenorhabditis-Elegans
RC Johnsen/Univ Missouri/Div Biol Sci/310 Tucker Hall/Columbia, MO 65211| --- From 10,900 F1 progeny of ethyl methanesulfonate (EMS)-mutagenized Caenorhabditis elegans nematodes, we isolated 194 lethal mutations on the left arm of LGV, a region balanced by the reciprocal translocation of eT1. The analysis of 166 of those mutations resulted in the identification of one deficiency and alleles of 78 genes including 38 new genes, thus increasing the number of identified essential genes to 101. We estimate that there are a minimum of 120 essential genes in this region, which comprises approximately 7% of the recombinational distance, although only about 4.2% of the genes, in C. elegans. We calculate that there are a minimum of 2850 essential genes in the genome. The left arm of LGV has two recombinational gene clusters separated by a high-recombination and/or essential gene-sparse region. One gene in this region, let-330, is the largest EMS target on the left arm of LGV, with twice as many alleles (16) as the next most EMS-mutable genes, let-332 and rol-3. Another gene in the sparse region, lin-40, and the region near lin-40 are major targets for Tc1 mobilization-induced mutagenesis. The analysis of essential genes in large regions should help to define C. elegans in terms of all its genes and aid in the understanding of the relationship of genome structure to genome function.
Kroeker, E.M.; Walker, V.K.
1991
Biochemi
Artic/En
L34/48
Developmental Expression and Hormonal Regulation of a Desiccation Stress Protein in Tenebrio molitor
VK Walker/Queens Univ/Dept Biol/Kingston K7L 3N6/Ontario, Canada| --- In a previous study it has been reported that dsp28 is induced during desiccation in Tenebrio larvae. During that study it was observed that in non-stressed larvae the concentration of dsp28 in hemolymph drops dramatically just prior to pupation. These results suggested that control of dsp28 synthesis is subject to environmental as well as hormonal cues. This study identifies a site of synthesis as fat body, as dsp28 was secreted into the medium during in vitro incubation of larval fat bodies. Using immunoelectrophoresis to determine protein concentration a developmental profile showing changes in levels of dsp28 in hemolymph during larval, pupal and adult stages of Tenebrio molitor was established. The concentration of dsp28 in larval hemolymph dropped from 4 to 5 mg/ml to 1.5 mg/ml just prior to pupation. This lower level was maintained until adults emerged when the concentration of dsp28 rose to prepupal levels again. Hormonal regulation is suggested since application of methoprene to newly-emerged pupae resulted in an increased incorporation of radiolabeled cysteine into dsp28.
RD Walter/Bernhard Nocht Inst Trop Med/Dept Biochem/Bernhard Nocht Str 74/D-2000 Hamburg 36, Fed Rep Ger| --- The occurrence of N-acetylglucosaminyltransferase, the initial step in the synthesis of the carbohydrate moiety of N-linked glycoproteins, is demonstrated in the microsomal fraction of the nematode Ascaris suum. Phosphatidylglycerol stimulated enzyme activity three- to six-fold without affecting the K(m) values of either substrates, uridinediphospho-N-acetylglucosamine or dolichylphosphate. The K(m) values were determined to be about 12-mu-M and 100-mu-g ml-1, respectively. The enzyme activity was strongly inhibited by tunicamycin acting as a competitive inhibitor with respect to the substrate uridinediphospho-N-glucosamine.
Hackstadt, T.
1991
MicrCell
Note/En
L34/47
Purification and N-Terminal Amino Acid Sequences of Chlamydia-Trachomatis Histone Analogs
J Bacteriol 173:7046-7049 (21)
NOV
OUTER-MEMBRANE PROTEIN; IDENTIFICATION; ENVELOPES
T Hackstadt/Niaid/Rocky Mt Labs/Intracellular Parasites Lab/Hamilton, MT 59840| --- DNA-binding proteins specific to Chlamydia trachomatis elementary bodies have been described and recently characterized as procaryotic histone analogs. I have developed an affinity purification procedure for the 18-kDa histone analog, Hc1, based on its affinity for polyanions. The availability of highly purified Hc1 has allowed for determination of its N-terminal amino acid sequence and should prove useful in studies of its biological function. The variable C. trachomatis histone analog not obtained by this procedure was electrophoresed onto Immobilon paper for sequencing. The N terminus of the variable histone was conserved among C. trachomatis serotypes L2, D, and B and was distinct from that of Hc1.
Kozak, M.
1991
Biochemi
Revie/En
L34/46
Structural Features in Eukaryotic Messenger RNAs That Modulate the Initiation of Translation
Molecular Cloning of the Mouse Mast Cell Protease-5 Gene - A Novel Secretory Granule Protease Expressed Early in the Differentiation of Serosal Mast Cells
HP Mcneil/Harvard Univ/Sch Med/Dept Med/Boston, MA 02115| --- cDNAs were isolated that encode mouse mast cell protease-5 (MMCP-5), an approximately 30,000 M(r) serine protease stored in the secretory granules of serosal mast cells (SMC) and Kirsten sarcoma virus-immortalized mast cells. Based on the deduced amino acid sequences of these cDNAs, MMCP-5 is synthesized as a 247-amino acid preproenzyme composed of a novel 19-residue hydrophobic signal peptide, a Gly-Glu activation peptide not present in other mast cell chymases, and a 226-amino acid protein that represents the mature enzyme. MMCP-5 possesses a unique Asn residue in the substrate binding cleft at residue 176 and is highly basically charged. The MMCP-5 gene was isolated, sequenced, and found to belong to a distinct subset of chymase genes. Allelic variations of the MMCP-5 gene were also detected. MMCP-5 is expressed in bone marrow-derived mast cells (BMMC), Kirsten sarcoma virus-immortalized mast cells, and SMC, but not in gastrointestinal mucosal mast cells of helminth-infected mice. The abundant levels of MMCP-5 mRNA in immature BMMC indicate that this chymase is expressed relatively early during the differentiation of mast cells. MMCP-5 is the first chymase to be molecularly cloned from progenitor mast cells and is also the first chymase shown to be expressed preferentially in the SMC subclass.
Robbi, M.; Beaufay, H.
1991
Biochemi
Artic/En
L34/46
The COOH Terminus of Several Liver Carboxylesterases Targets These Enzymes to the Lumen of the Endoplasmic Reticulum
M Robbi/Catholic Univ Louvain/Chim Physiol Lab/B-1200 Brussels, Belgium| --- To investigate the potential role of the COOH-terminal peptides in retaining a family of soluble carboxylesterases in the lumen of the endoplasmic reticulum, the pI 6.1 esterase cDNA has been cloned into the pKCR3 vector for transient expression in COS cells. The plasmid-encoded product appeared to be identical to the authentic enzyme: it was active on alpha-naphthyl acetate and behaved as a homotrimer of noncovalently bound 60-kDa subunits which contain a single, endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharide chain. This enzyme was retained in the transgenic COS cells. In contrast, a mutated form ending in HVER-COOH was secreted, indicating that the natural terminus HVEL-COOH contains topogenic information, with the ultimate Leu residue as an essential part. Variants of pI 6.1 esterase ending in HIEL-COOH, or HTEL-COOH were retained in cells to the same extent as the wild-type protein. Therefore, the sequences HIEL and HTEL present at the COOH termini of several liver esterases (rabbit forms 1 and 2, human esterase, mouse egasyn, and rat pI 6.4 esterase) most likely have a function in their localization in the endoplasmic reticulum. Finally, an HDEL-COOH variant of pI 6.1 esterase was also normally retained, demonstrating that this signal can be correctly decoded by the sorting machinery of mammalian cells. Cell retention signals of the type HXEL-COOH appear to be common in higher eukaryotes and tolerate considerable variation at the antepenultimate X residue.
Mcgovern, K.; Beckwith, J.
1991
Biochemi
Artic/En
L34/47
Membrane Insertion of the Escherichia-Coli MalF Protein in Cells with Impaired Secretion Machinery
J Beckwith/Harvard Univ/Sch Med/Dept Microbiol & Molec Genet/200 Longwood Ave/Boston, MA 02115| --- The MalF protein is an integral membrane protein of Escherichia coli containing eight membrane-spanning stretches and a large periplasmic domain of approximately 180 amino acids. We have asked whether this protein is dependent for its membrane insertion on the bacterial secretion machinery specified by the sec genes. Using azide to inhibit the SecA protein and sec mutants to reduce the functioning of the machinery, we have studied the membrane assembly of MalF and beta-galactosidase and alkaline phosphatase fusions to MalF. In no case did we see an effect of reducing sec gene function on the insertion of MalF or fusion proteins. Selection for mutants that would cause internalization of a MalF-beta-galactosidase hybrid protein yielded no mutations in sec genes. Our results suggest that MalF can assemble in the membrane independently of the bacterial secretion machinery.
Ellison, M.J.; Hochstrasser, M.
1991
Biochemi
Artic/En
L34/47
Epitope-Tagged Ubiquitin - A New Probe for Analyzing Ubiquitin Function
The Lamin B-Fold - Anti-Idiotypic Antibodies Reveal a Structural Complementarity Between Nuclear Lamin-B and Cytoplasmic Intermediate Filament Epitopes
SD Georgatos/European Molec Biol Lab/Program Cell Biol/Meyerhofstr 1/D-6900 Heidelberg, Fed Rep Ger| --- Previous studies have shown that nuclear lamin B binds specifically to the C-terminal domains of type III intermediate filament (IF) proteins under in vitro conditions. To further explore such site-specific interactions, we have used a two-step anti-idiotypic antibody approach. First, a monoclonal antibody disrupting the cytoplasmic IF network organization of living cells (mAb7A3) (Matteoni, R., and Kreis, T. E. (1987) J. Cell Biol. 105, 1253-1265) was characterized. Epitope mapping demonstrated that this antibody recognized a site located in the C-terminal domains of vimentin and peripherin (type III IF proteins). mAb7A3 was able to inhibit more than 80% of the in vitro binding of nuclear lamin B to PI, a synthetic peptide modeled after the C-terminal domain of peripherin that comprises a lamin B-binding site (Djabali, K., Portier, M. M., Gros, F., Blobel, G., and Georgatos, S. D. (1991) Cell 64, 109-121). In a second step, animals were immunized with mAb7A3 and the resulting anti-idiotypic sera were screened. Two of these antisera reacted specifically with nuclear lamin B but not with type A lamins or cytoplasmic IF proteins. The anti-lamin B activity of one of the antisera was isolated by affinity chromatography using a lamin B-agarose matrix. The reaction of these affinity-purified antibodies with lamin B was inhibited by mAb7A3. Furthermore, the anti-lamin B antibodies reacted with Fab fragments of mAb7A3 and abolished binding of lamin B to PI. From these data we conclude that anti-idiotypic antibodies against the paratope of mAb7A3 recognize specific epitopes of the lamin B molecule that have shapes complementary to the one of the C-terminal domain of type III IF proteins. We speculate that these (regional) conformations, which we term the "lamin B-fold," may also occur in non-lamin proteins that mediate the anchorage of IFs to various membranous organelles.
Selective Secretion of Azurophil Granule Contents Induced by Monovalent Cation Ionophores in Human Neutrophils - Evidence for Direct Ionophore Effects on the Granule Membrane
C Fittschen/Natl Jewish Ctr Immunol & Resp Med/Dept Pediat/1400 Jackson St/Denver, CO 80206| --- The study of factors contributing to secretion of neutrophil azurophil granules has previously been complicated by the inability to induce their release without concomitant exocytosis of specific granules. This publication describes the action of the first two agents, the Na-ionophore monensin and the K-ionophore nigericin which elicited only secretion of azurophil granules. Secretion depended on H+/alkali ion antiport by the ionophores since it was abolished in Na+- and K+-poor choline buffer. The secretagogue effects of both ionophores did not correlate with changes either in cytoplasmic pH or in transmembrane potential and were not associated with Ca-transients, but were closely associated with azurophil granule alkalinization suggesting that the secretory event resulted from alkali ion/H+ antiport at the granule membrane. Addition of zinc inhibited azurophil (but not specific) granule secretion in response to monensin, CB/FMLP, and zymosan, indicating that secretion induced by these agents shares a common step(s).
WH Muller/State Univ Utrecht/Dept Molec Cell Biol/Padualaan 8/3584 CH Utrecht, Netherlands| --- A combination of cryofixation without pre-treatment, freeze-substitution and low-temperature embedding was used to prepare specimens of Penicillium chrysogenum for electron microscopy. To produce specimens which are thin enough for appropriate cryofixation, the P. chrysogenum colonies were grown between dissected-dialysis tubing on an agar plate, which in addition allowed longitudinal sectioning. In contrast to classical chemical fixation, this preparation procedure resulted in excellent preservation of ultrastructure. Furthermore, the penicillin biosynthetic enzyme acyltransferase could be unequivocally located by immunogold labelling, indicating a preservation of antigenic properties of the specimen. Labelling density was not conspicuously affected when using different freeze-substitution media, but it was reduced after embedding in Epon 812.
Jones, J.T.; Gwynn, I.A.
1991
MicrCell
Artic/En
L34/47
A Method for Rapid Fixation and Dehydration of Nematode Tissue for Transmission Electron Microscopy
JT Jones/Univ Coll Aberystwyth/Dept Biol Sci/Aberystwyth SY23 1NE/Dyfed, Wales| --- A method is described using a microwave oven and acidified 2,2-dimethoxypropane to fix and dehydrate nematode tissue rapidly in preparation for transmission electron microscopy. Nematode tissue prepared using this method is well preserved compared with tissue fixed by conventional methods. The technique also enabled fixation of unhatched nematodes in the egg, a process that is almost impossible to achieve using conventional methods of fixation. Using the method described, differences were found between the cuticles of different nematode stages.
Tenter, A.M.; Zimmerman, G.L.; Johnson, A.M.
1991
ExpBioMe
Artic/En
L34/46
Separation of Antigens from Sarcocystis Species Using Chromatofocusing
AM Tenter/Tierarztlichen Hsch Hannover/Inst Parasitol/Bunteweg 17/D-3000 Hannover 71, Fed Rep Ger| --- Crude antigen preparations from bradyzoites of Sarcocystis species exhibit a high degree of cross-reactivity with antisera against heterologous Sarcocystis species, preventing the development of a species-specific immunological test for sarcocystiosis. In this study, we fractionated bradyzoite-derived protein extracts from Sarcocystis tenella, Sarcocystis arieticanis, Sarcocystis gigantea, and Sarcocystis muris by chromatofocusing and obtained distinct protein elution profiles for each species. We then examined the isolated protein fractions for antigenicity with homologous and heterologous reference sera in an enzyme-linked immunosorbent assay. Whereas some antigenic fractions of bradyzoite proteins had equally high reactivity with the homologous and heterologous sera, the reactivity of other fractions was 3-38 times higher with homologous serum than with heterologous sera. Mice immunized with less cross-reactive protein fractions of S. gigantea and S. muris bradyzoites produced a specific immune serum. Thus, it is possible to isolate species-specific antigens from crude mixtures of bradyzoite-derived Sarcocystis antigens for development of species-specific immunological tests for sarcocystiosis.
Xu, B.X.; Powell, M.R.
1991
ExpBioMe
Note/En
L34/46
Carbohydrate Epitopes Are Responsible for Antibody Cross-Reactivity in Trypanosoma-Cruzi-Infected Mice
J Parasitol 077: 808-810 (5)
OCT
STRAIN-INDUCED PROTECTION; C3H(HE) MICE
BX Xu/Ohio Univ/Dept Zool & Biomed Sci/Athens, OH 45701| --- Anti-Trypanosoma cruzi antibodies can be eluted from western blots of T. cruzi antigens and thereby are fractionated on the basis of the electrophoretic mobility of the antigens to which they bind. Antibodies fractionated by these methods can bind antigens with electrophoretic mobility different from those antigens from which they are eluted. Such antibodies thus are considered cross-reactive. Studies in which the target antigens are reacted with sodium periodate to destroy carbohydrate epitopes prior to exposure to the eluted antibodies revealed that antibodies are produced that bind to both carbohydrate and noncarbohydrate epitopes on western blots, but that most of the cross-reactive antibodies are directed toward carbohydrate moieties.
Grosskopf, D.G.; Felix, G.; Boller, T.
1991
AniPlaSc
Artic/En
L34/48
A Yeast-Derived Glycopeptide Elicitor and Chitosan or Digitonin Differentially Induce Ethylene Biosynthesis, Phenylalanine Ammonia-Lyase and Callose Formation in Suspension-Cultured Tomato Cells
DG Grosskopf/Friedrich Miescher Inst/Postfach 2543/CH-4002 Basel, Switzerland| --- An elicitor preparation consisting of glycopeptides partially purified from yeast extract and the membrane-perturbing agents chitosan and digitonin were compared with respect to their effects on ethylene biosynthesis, phenylalanine ammonia-lyase (PAL) activity and callose formation in tomato cells. The yeast-derived elicitor strongly induced ethylene biosynthesis and PAL activity. The two responses were closely correlated but independent since exogenous ethylene did not induce PAL and since cobalt ions, although completely inhibiting induction of ethylene biosynthesis, did not affect elicitor-dependent induction of PAL. The yeast-derived elicitor did not induce callose formation. Chitosan and digitonin, on the other hand, strongly induced callose formation and considerably induced PAL activity but stimulated ethylene biosynthesis only weakly. When these compounds were added in combination with yeast-derived elicitor, all responses were fully induced, indicating that none of the stimuli inhibited the response to the others. The induction of ethylene biosynthesis and PAL activity by the various stimuli was completely blocked by the protein kinase inhibitor, K-252a, while induction of callose formation was not inhibited.
Fischer, W.H.; Karr, D.; Jackson, B.; Park, M.; Vale, W.
1991
*CurBook
Artic/En
L34/48
Microsequence Analysis of Proteins Purified by Gel Electrophoresis --- In: Methods in Neurosciences, Vol 6 (Series: Methods in Neurosciences 6 (1991)), pp. 69-84. Editor(s): PM Conn. Academic Press Inc, 1250 6TH Ave, San Diego, CA 92101. ISBN 0-12-185262-8
Subcellular Distribution of Neuropeptides and Measurement of Their In Vitro Release --- In: Methods in Neurosciences, Vol 6 (Series: Methods in Neurosciences 6 (1991)), pp. 111-119. Editor(s): PM Conn. Academic Press Inc, 1250 6TH Ave, San Diego, CA 92101. ISBN 0-12-185262-8
Methods in Neurosciences, Vol 006: 111-119 ()
MC Beinfeld/St Louis Univ/Med Ctr/Dept Pharmacol & Physiol Sci/St Louis, MO 63104|
Jew, J.Y.; Fountain, N.B.; Jew, E.Y.
1991
*CurBook
Artic/En
L34/48
Ultrastructural Localization of Peptides - Comparison of Methods --- In: Methods in Neurosciences, Vol 6 (Series: Methods in Neurosciences 6 (1991)), pp. 130-147. Editor(s): PM Conn. Academic Press Inc, 1250 6TH Ave, San Diego, CA 92101. ISBN 0-12-185262-8
Methods in Neurosciences, Vol 006: 130-147 ()
JY Jew/Univ Iowa/Coll Med/Dept Anat/Iowa City, IA 52242|
Skidgel, R.A.
1991
*CurBook
Artic/En
L34/48
Assays for Arginine/Lysine Carboxypeptidases - Carboxypeptidases H (E-Enkephalin Convertase), Carboxypeptidases M, and Carboxypeptidases N --- In: Methods in Neurosciences, Vol 6 (Series: Methods in Neurosciences 6 (1991)), pp. 373-385. Editor(s): PM Conn. Academic Press Inc, 1250 6TH Ave, San Diego, CA 92101. ISBN 0-12-185262-8
Methods in Neurosciences, Vol 006: 373-385 ()
RA Skidgel/Univ Illinois/Coll Med/Dept Pharmacol/Peptide Res Lab/Chicago, IL 60680|
Highton, R.
1991
MolBiGen
Artic/En
L34/48
Molecular Phylogeny of Plethodonine Salamanders and Hylid Frogs - Statistical Analysis of Protein Comparisons
R Highton/Univ Maryland/Dept Zool/College Pk, MD 20742| --- The bootstrapping method of determining confidence in the topology of phylogenetic trees has been applied to electrophoretic protein data for two groups of amphibians: salamanders of two North American genera (Aneides and Plethodon) of the tribe Plethodontini and Holarctic hylid frogs. Some current methods of phylogenetic reconstruction for electrophoretic protein data have been evaluated by comparing the trees obtained from molecular data sets with available morphological data. Molecular data on the phylogenetic relationships of Aneides and Plethodon, data obtained from electrophoretic and immunological studies, indicate that Aneides probably was derived from western Plethodon subsequent to the separation of eastern and western Plethodon. Thus Plethodon very likely is a paraphyletic genus. The extremely low rate of morphological evolution in Plethodon compared with that in Aneides causes difficulty in indicating their evolutionary relationships taxonomically because there are no synapomorphic morphological characters that define either eastern or western Plethodon, whereas there are several for the genus Aneides. Thus molecular data alone probably indicate the evolutionary relationships of the species in these genera. Highton and Larson's (1979) arrangement of species of Plethodon into eight species groups is supported. The topologies of the unweighted pair-group method using arithmetic means (UPGMA) and distance Wagner trees were compared with independent morphological and molecular data on the relationships of the 28 plethodonine species. It was found that UPGMA trees indicate relationships that are more in agreement with other information than are those provided by distance Wagner trees. The use of the bootstrap technique indicates that the topologies of UPGMA trees are better supported statistically than are the topologies of distance Wagner trees. Moreover, different addition criteria produce a variety of distance Wagner trees with different topologies, each with several groupings that are not supported statistically. It is concluded that considerable caution should be used in interpreting the topology of distance Wagner trees. Very similar results were obtained with a second data set on 30 taxa of Holarctic hylid frogs. Trees obtained by the neighbor-joining method are more in agreement with UPGMA phenograms and other data, so this method of phylogenetic reconstruction may be useful to systematists not willing to assume constant rates of evolution. The suggested use, for transformation-series analysis, of electromorphs ordered by their electrophoretic mobility, is shown not to be a useful method for the analysis of genetic data. This is because there is not a significant correlation between albumin electrophoretic mobility and divergence, as measured by the quantitative immunological method of microcomplement fixation.
Normark, B.B.; Mccune, A.R.; Harrison, R.G.
1991
MolBiGen
Artic/En
L34/48
Phylogenetic Relationships of Neopterygian Fishes, Inferred from Mitochondrial DNA Sequences
BB Normark/Cornell Univ/Ecol & Systemat Sect/Corson Hall/Ithaca, NY 14853| --- To investigate the relationships among the three main groups of extant neopterygian fishes-Amiidae, Lepisosteidae, and Teleostei-we sequenced fragments of three mitochondrial genes from 12 different actinopterygian fishes and translated the nucleotide sequences into amino acid sequences. When all three regions are considered together, Amiidae clusters with Lepisosteidae in the most parsimonious cladograms, but other clades, such as Neopterygii and Teleostei, that are well supported by morphological evidence fail to emerge as monophyletic. When the cytochrome b sequences are analyzed together with previously published sequences for other taxa, the majority-rule consensus tree is consistent with the monophyly of Teleostei and Neopterygii and marginally supports the Amiidae+Lepisosteidae clade. In either analysis, when Neopterygii and Teleostei are constrained to monophyly, all the most-parsimonious cladograms support the Amiidae+Lepisosteidae topology. Where molecules and morphology disagree, provisional morphology-based constraints on the analysis of molecular data offer a practical means of integrating the two types of data.
Bulmer, M.
1991
MolBiGen
Artic/En
L34/48
Use of the Method of Generalized Least Squares in Reconstructing Phylogenies from Sequence Data
Mol Biol Evol 008: 868-883 (6)
NOV
Phylogenetic Reconstruction; Generalized Least Squares; Covariances of Branch Lengths; Phylogeny of Mammalian Orders; Human, Chimp, Gorilla Trichotomy; NUCLEOTIDE SUBSTITUTIONS; CONFIDENCE-LIMITS; STATISTICAL TEST; MOLECULAR-DATA; TREES; LIKELIHOOD; BOOTSTRAP; INFERENCE; EVOLUTION; DISTANCE
M Bulmer/Rutgers State Univ/Dept Biol Sci/Piscataway, NJ 08855| --- The method of generalized least squares provides a flexible method of phylogenetic reconstruction from sequence data, after reducing them to pairwise distances between species, corrected for multiple and back mutation. It gives efficient estimates of the branch lengths of a given tree. It also provides a natural measure of the departure of the observed from the predicted set of distances which has a chi-2 distribution under the true topology; this fact is used to construct a significance test on the topology and so to determine a "confidence interval" for the set of trees which are compatible with the data. To use this method it is necessary to know the variances and covariances of the corrected pairwise distances. A new method of estimating these variances and covariances empirically is described. The methodology is illustrated using data on the phylogeny of four mammalian orders (with the conclusion that rodents and lagomorphs are not sister groups) and of six primates (with the conclusion that the human/chimp/gorilla trichotomy cannot be resolved with these data alone).
Taq Polymerase; DNA Amplification; DNA Sequencing; DNA Cloning; DNA Polymorphism; Molecular Evolution; AQUATICUS DNA-POLYMERASE; GENETIC-VARIATION; AMPLIFICATION; CLONING; ERRORS
FJ Ayala/Univ Calif Irvine/Dept Ecol & Evolutionary Biol/Irvine, CA 92717|
Li, J.D.; Carroll, J.; Ellar, D.J.
1991
Multidis
Artic/En
L34/47
Crystal Structure of Insecticidal delta-Endotoxin from Bacillus thuringiensis at 2.5-A Resolution
DG Moerman/Univ British Columbia/Dept Zool/Vancouver V6T 2A9/BC, Canada| --- We have examined eight germline revertants generated by the excision of Tc1 from a site within the unc-22 gene of Caenorhabditis elegans. A rich variety of rearrangements accompanied Tc1 excision at this site, including transposon 'footprints', deletions of sequences flanking the insertion site and direct non-tandem duplications of flanking DNA. With only modest modification the double-strand gap repair model for transposition, recently proposed by Engles and co-workers (Cell 62: 515 - 525 1990), can explain even the most complex of these rearrangements. In light of this model rearrangements of the target site accompanying transposition/excision may not be the end result of imprecise excision of the element. Instead, these rearrangements may be the result of imprecise repair of the double-strand gap by the host replication and repair machinery. Sequences surrounding an insertion site influence the fidelity of gap repair by this machinery. This may lead to a number of possible resolutions of a double-strand gap as documented here for a Tc1 site in unc-22.
Hightower, R.; Baden, C.; Penzes, E.; Lund, P.; Dunsmuir, P.
1991
MolBiGen
Artic/En
L34/47
Expression of Antifreeze Proteins in Transgenic Plants
P Dunsmuir/DNA Plant Technol Corp/6701 San Pablo Ave/Oakland, CA 94608| --- The quality of frozen fruits and vegetables can be compromised by the damaging effects of ice crystal growth within the frozen tissue. Antifreeze proteins in the blood of some polar fishes have been shown to inhibit ice recrystallization at low concentrations. In order to determine whether expression of genes of this type confers improved freezing properties to plant tissue, we have produced transgenic tobacco and tomato plants which express genes encoding antifreeze proteins. The afa3 antifreeze gene was expressed at high steady-state mRNA levels in leaves from transformed plants, but we did not detect inhibition of ice recrystallization in tissue extracts. However, both mRNA and fusion proteins were detectable in transgenic tomato tissue containing a chimeric gene encoding a fusion protein between truncated staphylococcal protein A and antifreeze protein. Furthermore, ice recrystallization inhibition was detected in this transgenic tissue.
Robb, J.; Lee, S.W.; Mohan, R.; Kolattukudy, P.E.
1991
AniPlaSc
Artic/En
L34/47
Chemical Characterization of Stress-Induced Vascular Coating in Tomato
J Robb/Univ Guelph/Dept Molec Biol & Genet/Guelph N1G 2W1/Ontario, Canada| --- Indirect evidence suggests that vascular coatings formed by plants in response to stress consist of suberin-like substances containing lipid and phenolic compounds. To provide more direct chemical evidence that coatings are suberin, we used a natural pathogen, Verticillium albo-atrum, or a stress-responsive hormone, abscisic acid, to induce coating in two isolines of tomato (Lycopersicon esculentum L. cultivar Craigella) that are resistant or susceptible to the pathogen. Using treated petioles that had been monitored cytologically, chemical depolymerization followed by combined gas-liquid chromatography-mass spectrometry analysis of alkane-alpha,omega-diol levels confirmed the presence of suberin after induction of coating and showed quantitative differences between the isolines that correlated with cytological measurements of the coating response. Northern analysis of suberization-associated anionic peroxidase mRNA showed corresponding increases, and tissue blot analysis further indicated that induction of the mRNA was localized in the responding vascular bundles, as determined by suberin histochemistry. Taken together, these results provide chemical evidence that the coatings are mainly suberin.
Masuta, C.; Vandenbulcke, M.; Bauw, G.; Vanmontagu, M.; Caplan, A.B.
1991
AniPlaSc
Artic/En
L34/47
Differential Effects of Elicitors on the Viability of Rice Suspension Cells
M Vanmontagu/State Univ Ghent/Genet Lab/B-9000 Ghent, Belgium| --- We have compared the effects of two elicitors of defense-related processes on rice (Oryza sativa L.) suspension cells. Both chitosan and salicylic acid induced the accumulation of extracellular chitinase, thickening of the cell wall, and a variety of cytological changes in treated cells. Chitosan also induced the production of a brown pigment and cell death. Both of these effects depended on the availability of reactive oxygen species, because the damage was greatly reduced by either catalase or free-radical scavengers. Pretreating cells with salicylic acid also protected them from the cytotoxic effects of chitosan. This type of induced tolerance persisted when salicylic acid was removed and was not simply due to the release of extracellular substances, because salicylic acid-treated cells did not protect untreated cells from chitosan-induced death. Salicylic acid also stimulated the production of a 10-kilodalton subtilisin inhibitor that was not produced by chitosan-treated cells. Most of these changes are associated with the hypersensitive response of many plant species, including monocotyledons, and may serve as an in vitro model for investigating the biochemistry of some diseases.
Heitz, T.; Geoffroy, P.; Fritig, B.; Legrand, M.
1991
AniPlaSc
Artic/En
L34/47
2 Apoplastic alpha-Amylases Are Induced in Tobacco by Virus Infection
M Legrand/Univ Strasbourg 1/CNRS/Inst Biol Molec Plantes/12 Rue Gen Zimmer/F-67084 Strasbourg, France| --- Alpha-amylase activity (EC 3.2.1.1) is greatly increased in leaves of tobacco (Nicotiana tabacum L. cv Samsun NN) infected with tobacco mosaic virus (TMV). The kinetics of enzyme induction during the hypersensitive reaction resemble those of other hydrolases known to be pathogenesis-related proteins of tobacco. Two alpha-amylases were purified from TMV-infected leaves and shown to have features in common with well-characterized pathogenesis-related proteins: they are acidic monomers that can be separated upon electrophoresis on basic native gels, and they are found in the apoplastic compartment of the cell. This extracellular localization was demonstrated by comparing the alpha-amylase partition between the intercellular wash fluid and the cell extract with that of proteins of known cellular compartmentalization. These data indicate an active secretion of both alpha-amylases produced in tobacco upon TMV infection.
Pospieszny, H.; Chirkov, S.; Atabekov, J.
1991
AniPlaSc
Artic/En
L34/47
Induction of Antiviral Resistance in Plants by Chitosan
A 30-Residue-Long Export Initiation Domain Adjacent to the Signal Sequence Is Critical for Protein Translocation Across the Inner Membrane of Escherichia-Coli
H Andersson/Karolinska Inst/Ctr Biotechnol/Novum/Dept Molec Biol/S-14157 Huddinge, Sweden| --- Signal sequences serve to target proteins to the secretory pathway in both prokaryotic and eukaryotic cells. However, although necessary, the presence of a signal sequence is not always sufficient to ensure efficient membrane translocation. One feature of the nascent chain that adversely affects secretion, at least in Escherichia coli, is the presence of positively charged amino acids immediately downstream of the signal sequence. We have exploited this sensitivity to positively charged residues to demonstrate the presence of a sharply delimited "export initiation domain" that comprises the signal sequence and its almost-equal-to 30 downstream residues. A string of six consecutive lysines completely blocks translocation when placed inside this domain but not when placed only a few residues further away.
Michelmore, R.W.; Paran, I.; Kesseli, R.V.
1991
Multidis
Artic/En
L34/47
Identification of Markers Linked to Disease-Resistance Genes by Bulked Segregant Analysis - A Rapid Method to Detect Markers in Specific Genomic Regions by Using Segregating Populations
Proc Natl Acad Sci USA 088:9828-9832 (21)
NOV
Random Amplified Polymorphic DNA; Restriction Fragment Length Polymorphism; TOMATO; MAIZE; MAP
RW Michelmore/Univ Calif Davis/Dept Vegetable Crops/Davis, CA 95616|
Hodgkin, J.; Barnes, T.M.
1991
ExpBioMe
Artic/En
L34/47
More Is Not Better - Brood Size and Population Growth in a Self-Fertilizing Nematode
Proc R Soc Lond [Biol] 246: 19-24 (1315)
OCT 22
SEX-DETERMINATION GENE; CAENORHABDITIS-ELEGANS
J Hodgkin/MRC/Molec Biol Lab/Hills Rd/Cambridge CB2 2QH, England| --- The normal form of the nematode Caenorhabditis elegans is a self-fertilizing hermaphrodite, which produces from the same germ-line tissue first a limited number of sperm and then a larger number of oocytes. Self-progeny brood sizes are determined by the number of sperm, and most of the oocytes remain unfertilized. Therefore it might seem selectively advantageous to increase the number of sperm, and hence the size of the brood. A mutation that leads to a 50% increase in sperm production allows a comparison of population growth rates between the wild type (mean brood 327 progeny) and the mutant (mean brood 499 progeny). Wild-type populations grow faster, as measured by food consumption, indicating that increased brood size is not advantageous. The mutant appears to be at a disadvantage because the additional spermatogenesis leads to a delay in the onset of oogenesis, and hence to an increase in the minimum generation time. In support of the notion of an optimal brood size, it was found that different natural isolates of this species have self-fertilities similar to that of the standard laboratory strain, in the range 250-350 progeny per worm.
Hubbard, T.J.P.; Sander, C.
1991
Biochemi
Revie/En
L34/46
The Role of Heat-Shock and Chaperone Proteins in Protein Folding - Possible Molecular Mechanisms
Protein Eng 004: 711-717 (7)
OCT
Chaperone; Heat-Shock Proteins; Protein Engineering; Protein Folding; PURIFIED PRECURSOR PROTEIN; ESCHERICHIA-COLI; ATP HYDROLYSIS; UNCOATING ATPASE; MULTIGENE FAMILY; BINDING-PROTEINS; STRESS PROTEINS; DNA-REPLICATION; TRANSLOCATION; GROEL
TJP Hubbard/MRC/Cambridge Ctr Prot Engn/Hills Rd/Cambridge, England| --- Recently some heat-shock proteins have been linked to functions of 'chaperoning' protein folding in vivo. Here current experimental evidence is reviewed and possible requirements for such an activity are discussed. It is proposed that one mode of chaperone action is to actively unfold misfolded or badly aggregated proteins to a conformation from which they could refold spontaneously; that improperly folded proteins are recognized by excessive stretches of solvent-exposed backbone, rather than by exposed hydrophobic patches; and that the molecular mechanism for unfolding is either repeated binding and dissociation ('plucking') or translocation of the protein backbone through a binding cleft ('threading'), allowing the threaded chain to refold spontaneously. The observed hydrolysis of ATP would provide the energy for active unfolding. These hypotheses can be applied to both monomeric folding and oligomeric assembly and are sufficiently detailed to be open to directed experimental verification.
RJ Siezen/Netherlands Inst Dairy Res/Dept Biophys Chem/POB 20/6710 Ba Ede, Netherlands| --- Subtilases are members of the family of subtilisin-like serine proteases. Presently, > 50 subtilases are known, > 40 of which with their complete amino acid sequences. We have compared these sequences and the available three-dimensional structures (subtilisin BPN', subtilisin Carlsberg, thermitase and proteinase K). The mature enzymes contain up to 1775 residues, with N-terminal catalytic domains ranging from 268 to 511 residues, and signal and/or activation-peptides ranging from 27 to 280 residues. Several members contain C-terminal extensions, relative to the subtilisins, which display additional properties such as sequence repeats, processing sites and membrane anchor segments. Multiple sequence alignment of the N-terminal catalytic domains allows the definition of two main classes of subtilases. A structurally conserved framework of 191 core residues has been defined from a comparison of the four known three-dimensional structures. Eighteen of these core residues are highly conserved, nine of which are glycines. While the alpha-helix and beta-sheet secondary structure elements show considerable sequence homology, this is less so for peptide loops that connect the core secondary structure elements. These loops can vary in length by > 150 residues. While the core three-dimensional structure is conserved, insertions and deletions are preferentially confined to surface loops. From the known three-dimensional structures various predictions are made for the other subtilases concerning essential conserved residues, allowable amino acid substitutions, disulphide bonds, Ca2+-binding sites, substrate-binding site residues, ionic and aromatic interactions, proteolytically susceptible surface loops, etc. These predictions form a basis for protein engineering of members of the subtilase family, for which no three-dimensional structure is known.
Cagnon, C.; Valverde, V.; Masson, J.M.
1991
Biochemi
Artic/En
L34/46
A New Family of Sugar-Inducible Expression Vectors for Escherichia-Coli
