Biomedical subjects

D Yogev

Publications and source records attributed to D Yogev.

At least 37 records · Page 2Linked to original sources

PubMed1995

Increased structural and combinatorial diversity in an extended family of genes encoding Vlp surface proteins of Mycoplasma hyorhinis.

Variable lipoproteins (Vlp) constitute the major coat protein of Mycoplasma hyorhinis. They are products of multiple, divergent, single-copy genes organized in a chromosomal cluster. Three genes, vlpA, vlpB, and vlpC, have been previously identified in clonal isolates of M. hyorhinis SK76. Each is linked to a characteristic promoter region containing a homopolymeric tract of adenine residues [poly(A) tract], subject to hypermutation, that transcriptionally controls phase variation of vlp genes and leads to combinatorial surface mosaics of distinct Vlp products. The size of the natural vlp gene repertoire is unknown but may critically determine the degree of structural and combinatorial diversity available in this species. In this study, the vlp repertoire of M. hyorhinis GDL-1 was characterized and shown to contain three additional genes, vlpD, vlpE, and vlpF, clustered with other known vlp genes in the order 5'-vlpD-vlpE-vlpF-IS-vlpA-IS-vlpB-vlpC+ ++-3', where IS represents copies of the IS1221 element of M. hyorhinis. The 5' boundary of this expanded family was identical to that of the more limited family 5'-vlpA-IS-vlpB-vlpC-3' previously described in a clonal isolate of strain SK76. A recombinant construct containing vlpD, vlpE, and vlpF expressed antigenically distinguishable products corresponding to each gene. These genes encode characteristic C-terminal repetitive regions that are subject to size variation by insertion or deletion of intragenic repeats but maintain an extended, charged structure. Each vlp gene also contained characteristic alternative open reading frames, which provide a potential reservoir of coding sequence for Vlp diversity, possibly recruited through insertion and/or deletion mutations. These findings demonstrate a vastly expanded potential for structural diversity and combinatorial display of surface mosaics on this organism and suggest that modulation of the vlp repertoire, possibly in conjunction with mobile elements, may determine the capacity for surface variation in natural populations and laboratory strains of this mycoplasma species.

Amino Acid Sequence↗

PubMed1994

A surface epitope undergoing high-frequency phase variation is shared by Mycoplasma gallisepticum and Mycoplasma bovis.

We have recently reported that three distinct size- and phase-variable surface lipoproteins (Vsps) of the bovine pathogen Mycoplasma bovis possess a common epitope recognized by monoclonal antibody 1E5. In the present study, we show that this epitope is also present on a size-variant protein (PvpA) of the avian pathogen Mycoplasma gallisepticum. Application of monoclonal antibody 1E5 in Western immunoblot analysis of Triton X-114 phase-fractionated proteins and in colony immunoblots, as well as in trypsin and carboxypeptidase digestion experiments, has demonstrated that (i) PvpA is an integral membrane protein with a free C terminus, (ii) the shared epitope is surface exposed, and (iii) PvpA is subjected to high-frequency phase variation in expression. By using serum antibodies from M. gallisepticum-infected chickens, we were able to demonstrate the immunogenic nature of PvpA and identify three additional highly immunogenic Triton X-114 phase proteins (p67, p72, and p75) also undergoing high-frequency phase variation spontaneously and independently. Metabolic labeling experiments with [14C]palmitate and [14C]oleate revealed that PvpA, in contrast to p67, p72, and p75, is not lipid modified. Southern blot hybridization with restriction fragments carrying the pvpA gene of M. gallisepticum or the vspA gene of M. bovis against digested genomic DNA of the two Mycoplasma species indicated the absence of genetic relatedness between the pvpA and vspA genes. The apparent complexity of the antigenic variation phenomenon in M. gallisepticum is discussed.

Animals↗

PubMed1994

Antigen heterogeneity among isolates of Mycoplasma bovis is generated by high-frequency variation of diverse membrane surface proteins.

The protein and antigen profiles of 11 isolates of Mycoplasma bovis were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis of whole organisms. The isolates examined included the type strain PG45 and 10 other filter-cloned strains or purified isolates both from animals without clinical signs and from clinical cases of bovine mastitis, arthritis, or pneumonia. While the overall protein patterns visualized by silver staining were very similar, marked differences in the antigen banding profiles were detected by rabbit antiserum prepared against whole organisms from one of the strains analyzed. This antigenic heterogeneity was shown to be independent of the geographical origin, the type of clinical disease, and the site of isolation and was also observed among serial isolates from a single animal. Antigen profiles were further monitored throughout sequentially subcloned populations of the PG45 strain. This clonal analysis revealed a high-frequency variation in the expression levels of several prominent antigens. All of these variable antigens were defined by detergent-phase fractionation with Triton X-114 as amphiphilic integral membrane proteins. A subset of different-sized membrane proteins was identified by a monoclonal antibody raised against a PG45 subclone expressing a 63- and a 46-kDa variant antigen within that set. The selective susceptibility of these proteins to trypsin treatment of intact organisms and their ability to bind the monoclonal antibody in colony immunoblots demonstrated that they were exposed on the cell surface. In addition, their preferential recognition by serum antibodies from individual cattle with naturally induced M. bovis mastitis or arthritis confirmed that they were major immunogens of this organism. These studies establish that the apparent antigenic heterogeneity among M. bovis isolates reported here does not represent stable phenotypic strain differences generated from accumulated mutational events but reflects distinct expression patterns of diverse, highly variable membrane surface proteins.

Animals↗

PubMed1994

A family of phase- and size-variant membrane surface lipoprotein antigens (Vsps) of Mycoplasma bovis.

A set of strain- and size-variant highly immunogenic membrane surface protein antigens of Mycoplasma bovis, which has been identified by a monoclonal antibody, is shown in this report to make up a family of antigenically and structurally related lipid-modified proteins, designated Vsps (variable surface proteins). By systematic analysis of several isogenic clonal lineages of the type strain PG45, three members of this family have been identified, VspA, VspB, and VspC, each of which was shown to undergo independent high-frequency changes in size as well as noncoordinate phase variation between ON and OFF expression states. The monoclonal antibody-defined epitope common to VspA, VspB, and VspC was accessible on the cell surface in most, but not all, of the clonal populations analyzed and was present on a C-terminal limit tryptic fragment of each Vsp variant that was released from the membrane surface. VspA and VspC were distinguished from VspB by their selective detection with colloidal gold and by their distinctive reaction with a polyclonal antibody against M. bovis D490. VspA, VspB, and VspC were further distinguishable from one another by their characteristic patterns of degradation at carboxypeptidase Y pause sites. While these Vsp-specific structural fingerprints with an irregular periodic spacing were constant for similarly sized variants of a defined Vsp product, they showed distinct differences among variants differing in size. This variability included gain or loss of individual bands within distinct subsets of bands, as well as shifts of the entire banding patterns up- or downwards, indicating that insertions or deletions underlying Vsp size variation can occur at various locations either within the C-terminal domain or within other regions of these proteins. This was similarly confirmed by comparative epitope mapping analysis of tryptic cleavage products generated from different Vsp size variants. The Vsp family of M. bovis described in this study represents a newly discovered system of surface antigenic variation in mycoplasmas displaying features which closely resemble but are also different from the characteristics reported for the Vlp (variable lipoprotein) system of M. hyorhinis. The isogenic lineages established here provide key populations for subsequent analysis of corresponding genes to further elucidate Vsp structure and variation, which may have important relevance for a better understanding of the pathogenicity of this agent.

Animals↗

PubMed1993

Variation and genetic control of surface antigen expression in mycoplasmas: the Vlp system of Mycoplasma hyorhinis.

Surface antigenic diversity in the swine pathogen Mycoplasma hyorhinis is generated by random combinatorial expression and high-frequency phase variation of multiple, size-variant membrane surface lipoproteins (Vlps) which represent the major coat proteins of this wall-less procaryote. The distinctive structural basis for Vlp variation was revealed in a family of several related but divergent vlp genes. These occur in one cluster as single chromosomal copies, each encoding a conserved domain for membrane insertion and lipoprotein processing, and a divergent external domain that changes size by deletion or insertion of repetitive intragenic coding sequences while retaining a distinctive charge motif. Lack of detectable changes in restriction fragment patterns or DNA sequence of vlp structural genes during phase transitions between ON and OFF expression states ruled out long range genomic rearrangements and frameshift mutations as a means of controlling Vlp phase variation. However, highly homologous vlp promoter regions contain a homopolymeric tract of contiguous adenine residues [poly(A)] upstream of the transcriptional start site which is subject to frequent mutations altering its length. These mutations are the only sequence changes detected during phase transitions, and are highly correlated with the expression state of each vlp gene. This suggests a mechanism of transcriptional control regulating Vlp phase variation by critical changes within the poly(A) region affecting the spacing between the -10 and -35 hexamers or a putative regulator binding site. The multiple levels of structural and antigenic diversity embodied in the vlp gene family may provide essential adaptive capabilities for this wall-less microbial pathogen.

Animals↗

PubMed1993

Antigenic variation in Mycoplasma hyorhinis: increased repertoire of variable lipoproteins expanding surface diversity and structural complexity.

VlpE is characterized as a new member in a family of variable surface lipoproteins (Vlps) of Mycoplasma hyorhinis. VlpE shows phenotypic variation in expression and size within isogenic lineages of some strains but is absent from lineages of other strains that express only three previously known Vlps. Expression of four Vlps in some cells further indicates the presence and usage of an expanded reservoir of Vlp coding sequences, which greatly increases the capacity for surface diversification.

Antibodies, Monoclonal↗

PubMed1992

Electric-field-induced reconnection of severed axons.

We report the first successful axon reconnection in the earthworm (Lumbricus terrestris) medial giant axon (MGA) by electric fields generated by electrical pulses of 10-100 microseconds duration and 80-200 V amplitude. Reconnection was documented by light and electron microscopy, and by transport of Lucifer yellow dye across the reconnected MGA segments. Direct repair of a severed nerve axon promises the advantages of preserving axon viability and distal connections.

Animals↗

PubMed1991

Laser-induced reconnection of severed axons.

After their axons have been severed, nerve cells can achieve functional recovery either by regrowth of the injured cells or by direct repair of the injured cell at the site of injury. Direct repair of a severed axon promises the advantages of preserving the viability and existing connections of the axon distal to the injury. We report here the first successful axon reconnection in the earthworm (Lumbricus terrestris) medial giant axon (MGA) in vitro system (1) by the application of well-focused 15 nsec, 5 to 50 muJ/pulse, 308 nm laser pulses. Axon reconnection is documented by light and electron microscopy, as well as by transfer of the iontophoretically injected fluorescent dye, Lucifer Yellow, across the reconnected MGA segments.

Animals↗

PubMed1991

Molecular basis of Mycoplasma surface antigenic variation: a novel set of divergent genes undergo spontaneous mutation of periodic coding regions and 5' regulatory sequences.

Antigenic diversity is generated in the wall-less pathogen Mycoplasma hyorhinis by combinatorial expression and phase variation of multiple, size-variant membrane surface lipoproteins (Vlps). The unusual structural basis for Vlp variation was revealed in a cluster of related but divergent vlp genes, vlpA, vlpB and vlpC, which occur as single chromosomal copies. These encode conserved N-terminal domains for membrane insertion and lipoprotein processing, but divergent external domains undergoing size variation by loss or gain of repetitive intragenic coding sequences while retaining a motif with distinctive charge distribution. Genetic analysis of phenotypically switched isogenic lineages representing ON or OFF expression states of Vlp products ruled out chromosomal rearrangement or frameshift mutations as mechanisms for Vlp phase variation. However, highly conserved vlp promoter regions contain a tract of contiguous A residues immediately upstream of the -10 box which is subject to frequent mutations altering its length in exact correspondence with the ON and OFF phase states of specific genes. This suggests a mechanism of transcriptional control regulating high frequency phase variation and random combinatorial expression of Vlps. The multiple levels of diversity embodied in the vlp gene cluster represents a novel adaptive capability particularly suited for this class of wall-less microbe.

Amino Acid Sequence↗

PubMed1991

Sequence and TnphoA analysis of a Mycoplasma hyorhinis protein with membrane export function.

Proteins translocated across the single plasma membrane of mycoplasmas (class Mollicutes) represent important components likely to affect several interactions of these wall-less microbes with their respective hosts. However, identification and functional analysis of such proteins is hampered by the lack of mutational systems in mycoplasmas and by a perceived limitation in translating recombinant mycoplasma genes containing UGA (Trp) codons in other eubacteria. Here we directly analyze a gene encoding a Mycoplasma hyorhinis protein capable of promoting its membrane translocation. It was initially detected by screening a recombinant phage genomic library with antibody from a host with M. hyorhinis-induced arthritis and was localized by Tn5 and deletion mutations affecting expression of antigenic translational products. Sequence analysis of the isolated gene predicted a hydrophilic protein, P101, containing three UGA codons and a putative signal peptide with an uncharacteristic cluster of positively charged amino acids near its C terminus. Nevertheless, lambda::TnphoA transposon mutagenesis of an Escherichia coli plasmid bearing the p101 gene resulted in p101::TnphoA fusions expressing products that could translocate as much as 48 kDa of the P101 sequence (up to the first UGA codon) across the E. coli plasma membrane. Fusion proteins containing mature P101 sequences expressed mycoplasma epitopes and were found by cell fractionation and detergent phase partitioning to be integral membrane proteins in E. coli, suggesting a lack of signal peptide cleavage in this system. Importantly, identification of P101 by direct analysis of its export function relied neither on prior identification of the mycoplasmal product nor on complete expression of the product from the cloned mycoplasma gene.

Amino Acid Sequence↗

PubMed1990

Nucleotide sequence and codon usage of the elongation factor Tu(EF-Tu) gene from Mycoplasma pneumoniae.

The Mycoplasma pneumoniae tuf gene, encoding the elongation factor protein Tu, was cloned and sequenced. The nucleotide sequence of the mycoplasmal gene showed about 60% homology to the sequences of tuf genes of other prokaryotes, yeast mitochondria and Euglena gracilis chloroplasts, and about 75% similarity was found when comparing the deduced amino acid sequences of the various Tu proteins. The relatively low G + C content (40%) of the M. pneumoniae DNA was reflected in a low G + C content (44.6%) of the tuf gene, and in a preferential use of adenine and uracil at the third position of codons, yet codon usage analysis revealed the presence of almost all of the codons of the genetic code in the mycoplasmal gene. Southern blot hybridization of digested DNAs of 11 Mollicutes species with the entire M. pneumoniae tuf gene and with its 5' part suggested the presence of one copy only of this gene in the representative species of the Mollicutes. In this respect, the Mollicutes resemble Gram-positive bacteria and differ from the Gram-negative bacteria, which carry two copies of the tuf gene.

Amino Acid Sequence↗

PubMed1989

DNA probes for Mycoplasma gallisepticum and Mycoplasma synoviae: application in experimentally infected chickens.

DNA probes specific for Mycoplasma gallisepticum and M. synoviae were selected from genomic libraries prepared in the pUC13 vector. The probes hybridized with the DNA of a wide spectrum of strains within each homologous species, but did not react with the heterologous species or with DNA from any other avian mycoplasma or bacteria tested. Experimental infection and contact exposure of chickens to M. gallisepticum served as models to test the effectiveness of the DNA probe in diagnosis as compared with serological and culture detection methods carried out in parallel. A correlation was generally found between the level of M. gallisepticum in tracheal swabs and the effectiveness of the probe, although a predictably reactive level of mycoplasmas was not always detected. Treatment of clinical specimens with acetylcysteine to disrupt mucus improved the detection rate. Dot-blot hybridization with probe pMG4 enabled positive identification of M. gallisepticum at an early stage of infection, prior to the development of a serological response in the infected chicken. Results are obtainable within 4 days of sampling, much more rapidly than culture, and also in clinical specimens from which mycoplasma isolation is impossible, such as carcasses. The results indicate that the use of DNA probes for the early and rapid detection of M. gallisepticum infection is feasible; a development which can replace laborious culture techniques and less effective serological methods, and thus reduce the time required for diagnosis.

Animals↗

PubMed1989

Genetic and antigenic relatedness between Mycoplasma gallisepticum and Mycoplasma synoviae.

The two avian pathogens Mycoplasma gallisepticum and Mycoplasma synoviae were found, by Southern blot hybridization of their digested DNAs, to share genomic nucleotide sequences additional to those of the highly conserved ribosomal RNA genes. The assumption that some of the shared sequences encode for antigens or epitopes common to both mycoplasmas was supported by Western immunoblot analysis of cell proteins of one mycoplasma with specific antiserum to the other mycoplasma. Interestingly, the band patterns of reactive antigens were different for some of the M. gallisepticum strains, supporting the concept that the species is genotypically variable. The results of the present study may explain the cross-reactivity of the two mycoplasmas noted previously in a variety of routine serological tests.

Animals↗

PubMed1989

Duplication of the tuf gene: a new insight into the phylogeny of eubacteria.

The conservation and duplication of the tuf gene encoding the elongation factor EF-Tu were used to define phylogenetic relationships among eubacteria. When the tufA gene of Escherichia coli was used as a probe in hybridization experiments, duplicate tuf genes were found in gram-negative bacteria from three major phyla: purple bacteria, bacteroides, and cyanobacteria. Only a single copy of tuf was found in gram-positive bacteria, including mycobacteria and mycoplasmas. Gram-positive clostridia were found to carry two copies of tuf.

Bacteroides↗

PubMed1988

Distinction of species and strains of mycoplasmas (mollicutes) by genomic DNA fingerprints with an rRNA gene probe.

Genomic fingerprints of Acholeplasma laidlawii, Mycoplasma hominis, and Mycoplasma pneumoniae strains were obtained by Southern blot hybridization of the digested mycoplasmal DNAs with an rRNA gene probe. The hybridization patterns revealed genotypic heterogeneity among A. laidlawii and M. hominis strains and a remarkable degree of homogeneity among M. pneumoniae strains isolated from pneumonia patients during a 10-year period. Genomic fingerprints with the rRNA gene probe can thus serve as indicators of intraspecies genetic homogeneity or heterogeneity and can provide a new, sensitive tool for strain identification with a potential for application in epidemiology.

DNA, Bacterial↗

PubMed1987

DNA probes for detection and identification of Mycoplasma pneumoniae and Mycoplasma genitalium.

DNA probes specific for Mycoplasma pneumoniae and Mycoplasma genitalium were selected from genomic libraries prepared in pUC13. The 32P-labeled probes could detect, by dot blot hybridization, down to about 0.1 ng of the specific mycoplasma DNA or 10(5) CFU. Biotinylation of probe decreased the sensitivity of detection and produced nonspecific background reactions with nonhomologous DNAs. Sulfonation of probe yielded a similar level of sensitivity with less background.

DNA, Bacterial↗

PubMed1987

DNA probes for detection and identification of mycoplasmas (Mollicutes).

DNA probes are expected to prove a specific, sensitive, rapid and inexpensive means for diagnosis of mycoplasma infections, replacing procedures that depend on cultivation of the fastidious organisms. Probes made up of conserved genes, such as rRNA genes, do offer the advantage of identifying and distinguishing multiple species with a single labeled reagent. The mycoplasmal rRNA gene probe pMC5 was effective in detection and identification of mycoplasmas infecting cell cultures. However, use of pMC5 for detection of spiroplasmas and mycoplasma-like organisms (MLOs) in infected plants was hindered by hybridization of this probe with chloroplast rRNA genes. Moreover, for identifying species and strains by pMC5, a complex hybridization procedure--involving DNA purification, digestion, electrophoresis, and Southern blot hybridization--is required. More specific DNA probes, on the other hand, can identify specific Mollicutes by the much simpler, faster and more sensitive dot blot technique. Thus, a probe made of a cloned Spiroplasma citri plasmid could detect by this technique as little as 10 pg of S. citri DNA (equivalent to about 10(3) organisms) in infected plants and insects. DNA probes specific for Mycoplasma pneumoniae and M. genitalium were selected from genomic libraries and prepared in pUC13 by screening the libraries for inserts hybridizing only with DNA of the specific mycoplasma. The probes, labeled by nick translation with 32P-nucleotides, could detect as little as approximately 100 pg of the specific mycoplasmal DNA by dot blot hybridization. To eliminate radioactivity, the above DNA probes were labeled by biotinylation of sulfonation systems. Dot blot hybridization with these probes showed decreased sensitivity of detection by about one order of magnitude, and some nonspecific background reaction with large quantities of nonhomologous DNAs.

Animals↗

PubMedDate not supplied

Genetic relatedness among Ureaplasma urealyticum serotypes (serovars).

Fourteen serotypes (serovars) are currently recognized in the species Ureaplasma urealyticum. DNA-DNA hybridization tests, cleavage of genomic DNA by restriction endonucleases, and polyacrylamide gel electrophoresis of cell proteins indicate that the U. urealyticum serotypes fall into two genotypically distinct, but related, clusters. Southern hybridization tests of mycoplasmal ribosomal RNA gene probes with DNAs of the various serotypes digested by restriction endonucleases showed that the U. urealyticum chromosome carries two sets (operons) of ribosomal RNA genes. However, the hybridization patterns produced by the serotypes showed differences which were most pronounced among serotypes belonging to the two different clusters.

Electrophoresis, Polyacrylamide Gel↗