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At least 1,135 records · Page 63Linked to original sources
PubMed1993

In vitro production of toxin-coregulated pili by Vibrio cholerae El Tor.

Toxin-coregulated pili (TCP) have been shown to be a virulence determinant and protective antigen for Vibrio cholerae strains of classical biotype, but their role in infection by strains of the alternative El Tor biotype remains uncertain. In an attempt to demonstrate TCP production by El Tor vibrios we have over-expressed the El Tor tcpA gene in Escherichia coli, in order to prepare a biotype-specific anti-TcpA serum. This reagent proved to be a very sensitive indicator of TcpA production in immunoblotting studies, but failed to detect polymerized pili on the bacterial surface by immuno-electron microscopy (IEM). However, results with an analogous reagent which detects classical TcpA suggested that antisera to unprocessed TcpA do not efficiently recognize epitopes on native proteins. Accordingly we prepared a serum against a cell envelope fraction rich in processed El Tor TcpA. After extensive absorption this reagent reacted almost exclusively with TcpA by immunoblotting; when used in IEM, it allowed visualization of typical TCP bundles on the surfaces of each of five El Tor strains known to produce TcpA in vitro. We have previously reported that the El Tor strain O17 does not synthesize TcpA during growth in vitro, but that an O17 clone carrying a cosmid of classical origin expresses surface TCP. Using the biotype-specific anti-TcpA reagents in immunoblotting studies it has been possible to detect the product of the host chromosomal tcpA gene in such constructs; transcription of this gene was confirmed using biotype-specific tcpA probes. IEM revealed that El Tor TcpA was present in the TCP bundles associated with the O17 cosmid clones. Further studies suggest that regulation of the genes encoding TcpA and cholera toxin varies between different strains of El Tor biotype.

Antibodies, Bacterial↗
PubMed1997

Genetic and physical maps of the Alcaligenes eutrophus CH34 megaplasmid pMOL28 and its derivative pMOL50 obtained after temperature-induced mutagenesis and mortality.

We describe the construction of restriction and genetic maps of plasmid pMOL28, which has a size of approximately 180 kb. To do so, partial BamHI-digested DNA of pMOL28 was cloned into cosmid pLAFR3, which can package up to 20-30 kb inserted DNA. Subsequently, a cosmid walking strategy, combined with BamHI or EcoRI restriction analysis and hybridization, was used to construct the restriction maps for both enzymes. On these maps, 35 BamHI fragments and 29 EcoRI fragments were placed, accounting for a total size of approximately 180 kb. We also analyzed several rearranged derivatives of pMOL28 that were obtained after a process of temperature-induced mortality and mutagenesis (TIMM), which is characteristic for Alcaligenes eutrophus CH34 and related strains. The restriction and genetic maps of pMOL50 (222 kb), an enlarged derivative of pMOL28 obtained after TIMM, were constructed. By comparing the pMOL28 and pMOL50 maps, at least two transposable elements were identified which participated in the formation of pMOL50 from pMOL28 during TIMM. These transposable elements were IS1086, which was recently sequenced, and a new element named IS1089, which is located on the 44-kb inserted DNA fragment in pMOL50. Partial sequencing of IS1089 revealed similarity of this element with IS1071 of the chlorobenzoate catabolic transposon Tn5271 of Alcaligenes sp. BR60.

Alcaligenes↗
PubMed1997

Cloning, overexpression, and mutagenesis of the gene for homoprotocatechuate 2,3-dioxygenase from Brevibacterium fuscum.

Homoprotocatechuate (hpca, 3,4-dihydroxyphenylacetate) is a central intermediate for the bacterial degradation of aromatic compounds. Homoprotocatechuate 2,3-dioxygenase (HPCD) catalyzes the key ring cleavage step in the metabolism of hpca by the Gram (+) bacterium Brevibacterium fuscum to yield alpha-hydroxy-delta-carboxymethyl cis-muconic semialdehyde. A genomic DNA library of B. fuscum was constructed in Escherichia coli using a cosmid vector and screened by spraying the cells with hpca. One clone was found to contain the gene for HPCD based on its ability to convert hpca into the yellow-colored product. This cosmid clone was further subcloned and the gene for HPCD was localized and sequenced. The open reading frame codes for a protein with 365 amino acids and M(r) = 41,699, in accord with the characteristics of the previously purified wild-type enzyme. The gene for HPCD was overexpressed in E. coli to approximately 30% of the total soluble protein, and purification of the recombinant enzyme to apparent homogeneity was achieved by a two-step procedure. Iron was the only abundant metal found in the purified recombinant enzyme, and the specific activity per iron was comparable to that observed for the wild-type enzyme. The deduced amino acid sequence of HPCD has a very high level of homology (78.6% identity in the 337-aa overlap) to the manganese-dependent homoprotocatechuate 2,3-dioxygenase (MndD) from Arthrobacter globiformis CM-2. The basis for the difference in metal selection by HPCD and MndD was investigated by mutagenesis of a 50-base-pair region of the HPCD gene containing three frame shifts relative to the MndD gene. The purified triple mutant of HPCD did not exhibit a significant change in the metal content; therefore, other factors must contribute to the selection of the active site metal.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1993

Identification of genomic regions of the herpesvirus of turkeys (HVT) with helper activity for avian adeno-associated virus (AAAV).

Herpesvirus of turkeys (HVT) is a potent helper for the defective parvovirus avian adeno-associated virus (AAAV). To study the helper mechanism at the molecular level, we established a complete cosmid library of HVT DNA in a set of seven overlapping clones and transiently cotransfected secondary chicken embryo fibroblast (CEF) cells with AAAV DNA and recombinant cosmids (cBL) (individual as well as in different combinations). Using an AAAV-specific indirect immunofluorescence assay, we identified four regions on the HVT genome, represented by cBL267, cBL27, cBL33, and cBL34, which express helper functions for AAAV. As demonstrated by infection studies with extracts from cotransfected CEF cells, cBL267 promotes productive AAAV growth, while the helper effect induced by cBL27, cBL33, and cBL34 is limited to the synthesis of noninfectious AAAV antigen. In view of the data presented, possible HVT-specific helper mechanisms for AAAV are discussed.

Animals↗
PubMed1994

Gene isolation through genomic complementation using an indexed library of Chlamydomonas reinhardtii DNA.

Hundreds of mutants with defects in a variety of physiologically important functions, such as photosynthesis, respiration, flagellar motility, phototaxis, circadian rhythms and the cell cycle, have been isolated from cultures of Chlamydomonas reinhardtii. In only a few cases have the genes responsible for these mutations been cloned and sequenced. The development of efficient methods for transformation with nuclear genes [7] has allowed the recent demonstration of gene isolation through genomic complementation with a pooled library of C. reinhardtii DNA [9]. To improve the efficiency with which genes complementing a particular mutation can be isolated, we have established an indexed (ordered) cosmid library of 11,280 individual clones contained in the separate wells of 120 microtiter plates. The average insert size is ca. 38 kb. PCR analysis of five sequenced nuclear genes present in the Chlamydomonas library revealed a range from two copies for the alpha 2- and beta 2-tubulin genes to at least seven copies for the argininosuccinate lyase gene. Overall, these five clones were represented an average of > or = 3.4 times in the library. Thus, the probability that any one particular nuclear gene of < 1000 bp will be found in the library is > or = 97%, and the probability that a gene of ca. 10,000 bp will be found in the library is ca. 92%. Rapid screening methods with cosmid DNAs pooled from individual microtiter dishes have been applied successfully. Bacteria containing clones of the argininosuccinate lyase gene have been identified through genomic complementation of a Chlamydomonas mutant bearing an inactive argininosuccinate lyase gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Structural analysis of the mouse T-cell receptor Tcra V2 subfamily.

Cosmid clones containing T-cell receptor Tcra V2 subfamily gene segments have been isolated from a BALB/c cosmid library and subjected to DNA sequence analysis. The V gene segments in the Tcra V2 subfamily differ from each other by 3%-7% at the nucleotide level and 5%-16% at the amino acid level. T-cell receptor Tcra V2 gene segment polymorphisms have been identified in the B10.PL and PL/J mouse strains with a Tcra V2 subfamily-specific probe. These V gene segment polymorphisms may cause the differential Tcra V gene usage in induced experimental allergic encephalomyelitis between B10.PL and PL/J mice.

Amino Acid Sequence↗
PubMed1994

Multiple TL-like loci in the grc-G/C region of the rat.

The grc-G/C region of the rat is homologous to the Q/TL region of the mouse, and deletions in this region are associated with fetal mortality, developmental defects, and decreased resistance to cancer. Several cosmids spanning approximately 45 kilobases of this region were analyzed for their class I loci, using a mouse general class I probe (pAG64c), grc-specific probes (pGRC1.4, pGRC1.7), and four probes derived from the TL-like locus RT1.N1. The results showed that TL-like genes other than RT1.N1 exist in the rat: a duplicated gene, RT1.N2, was identified, sequenced, and shown to be 99.3% similar to RT1.N1; and a third TL-like gene, RT1.N3, was isolated from a cDNA library, sequenced, and shown to be 92.8% similar to RT1.N1. These observations suggest that the rat TL-like loci are duplicated and that there is more than one cluster of these duplicated genes. The TL-like genes are transcribed predominantly in the thymus, except in grc- strains, and their level of transcription increases during fetal life and reaches its maximum at birth. Finally, a cosmid that appears to identify the end of the deletion in grc- strains was identified.

Amino Acid Sequence↗
PubMed1994

Mapping of two new markers within the smallest interval harboring the spinal muscular atrophy locus by family and radiation hybrid analysis.

The locus responsible for the childhood-onset proximal spinal muscular atrophies (SMA) has recently been mapped to an area of 2-3 Mb in the region q12-q13.3 of chromosome 5. We have used a series of radiation hybrids (RHs) containing distinct parts of the SMA region as defined by reference markers. A cosmid library was constructed from one RH. Thirteen clones were isolated and five of these were mapped within the SMA region. Both RH mapping and fluorescence in situ hybridization analysis showed that two clones map in the region between loci D5S125 and D5S351. One of the cosmids contains expressed sequences. Polymorphic dinucleotide repeats were identified in both clones and used for segregation analysis of key recombinant SMA families. One recombination between the SMA locus and the new marker 9Ic (D5S685) indicates that 9Ic is probably the closest distal marker. The absence of recombination between the SMA locus and marker Fc (D5S684) suggests that Fc is located close to the disease gene. These new loci should refine linkage analysis in SMA family studies and may facilitate the isolation of the disease gene.

Animals↗
PubMed1993

Isolation and mapping of discrete DXS101 loci in Xq22 near the X-linked agammaglobulinaemia gene locus.

The X-linked agammaglobulinaemia (XLA) gene locus has previously been mapped to Xq22 in genetic linkage studies. The DXS101 locus has shown no recombinations with XLA in the ten informative meioses investigated so far. The DXS101 sequence, recognised by the cX52.5 plasmid, is moderately repeated in Xq22. We have isolated cosmids which contain this sequence; two copies of which have been found to lie near DXS178 and XLA, and a third copy which lies near the PLP gene, distal to these loci. We have used the cosmids to generate probes which should be of use for RFLP analysis, and thus in both prenatal diagnosis and carrier testing for XLA, and in constructing a genetic map of this region. These probes will also be used to complement the genetic map in the construction of a complete physical map of Xq22.

Agammaglobulinemia↗
PubMed1995

Positional mapping of loci in the DiGeorge critical region at chromosome 22q11 using a new marker (D22S183).

The majority of patients with DiGeorge syndrome (DGS) and velo-cardio-facial syndrome (VCFS) and a minority of patients with non-syndromic conotruncal heart defects are hemizygous for a region of chromosome 22q11. The chromosomal region that is commonly deleted is larger than 2 Mb. It has not been possible to narrow the smallest region of overlap (SRO) of the deletions to less than ca 500 kb, which suggests that DGS/VCFS might be a contiguous gene syndrome. The saturation cloning of the SRO is being carried out, and one gene (TUPLE1) has been identified. By using a cosmid probe (M51) and fluorescence in situ hybridization, we show here that the anonymous DNA marker locus D22S183 is within the SRO, between TUPLE1 and D22S75 (probe N25). A second locus with weak homology to D22S183, recognized by cosmid M56, lies immediately outside the common SRO of the DGS and VCFS deletions, but inside the SRO of the DGS deletions. D22S183 sequences are strongly conserved in primates and weaker hybridizing signals are found in DNA of other mammalian species; no transcripts are however detected in polyA+ RNA from various adult human organs. Probe M51 allows fast reliable screening for 22q11 deletions using fluorescence in situ hybridization. A deletion was found in 11 out of 12 DGS patients and in 3 out of 7 VCFS patients. Two patients inherited the deletion from a parent with mild (atypical) symptoms.

Abnormalities, Multiple↗
PubMed1993

Structure and expression analysis of a member of the human glutamate dehydrogenase (GLUD) gene family mapped to chromosome 10p11.2.

Glutamate dehydrogenase (GLUD) is a key metabolic enzyme of the mitochondrion, playing an important role in mammalian neuronal transmission. GLUD deficiency has been associated with certain forms of neurodegeneration in the human cerebellum. Genomic DNA blot hybridization analysis and identification of a large number of GLUD-specific genomic clones have suggested that human GLUD is encoded by a multigene family consisting of at least six members. A functional GLUD gene, GLUD1, has been mapped to chromosome 10q22.3-23 and a full-length "processed" GLUD gene, GLUDP1, to chromosome Xq22-23. In the context of studing the structure, the role, and the chromosomal organization of the other family members, we have analysed in detail, a cosmid clone solely reactive with the 3' region of the GLUD cDNA. Structure and expression analysis of its GLUD-specific region suggests that it represents a truncated "processed" GLUD pseudogene. Fluorescence in situ hybridization using the entire cosmid as a probe, mapped this GLUD gene locus, termed GLUDP5, to chromosome 10p11.2.

Amino Acid Sequence↗
PubMed1994

Ordering markers in the region of the ataxia-telangiectasia gene (11q22-q23) by fluorescence in situ hybridization (FISH) to interphase nuclei.

Fluorescence in situ hybridization (FISH) to interphase nuclei was performed to order probes corresponding to bands 11q22-q23 where the ataxia-telangiectasia (AT) gene(s) have been located. Cosmid probes and one phage probe previously localized to this chromosome 11 region by FISH to metaphase chromosomes, were hybridized to interphase nuclei of the somatic cell hybrid J1a, which contains chromosome 11 as the only human chromosome. Two-color FISH was used with a centromeric reference probe marker. The following order was obtained: cen-D11S385 (CJ52.75)-CJ52.3-D11S384 (CJ52.193)-CJ52.114-D11S424 (CJ52.77)-D11S132-NCAM-D11S351 (CJ52.208)-tel. The validity of using the centromeric probe was illustrated by showing that a probe corresponding to 11p13 hybridized more closely to the centromere than a probe corresponding to 11q22-q23, and by using cosmids hybridized three by three.

Ataxia Telangiectasia↗
PubMed1992

A non-alphoid repetitive DNA sequence from human chromosome 21.

A non-alphoid repetitive DNA from human chromosome 22, consisting of a 48-bp motif, shows homology to both G-group chromosomes in the gorilla, thus indicating the presence of additional repeat family members on further human chromosomes. Therefore, we screened a chromosome-21-specific cosmid library using this repetitive sequence from chromosome 22 (D22Z3). Some 40-50 cosmid clones were positive in tests for hybridization. One of the clones giving the strongest signals was digested with EcoRI/PstI, which we knew to cut frequently within the repeats; this resulted in fragments containing repeat units only. The fragments were subcloned into plasmid vector pTZ 19. Sequence-analysis of a 500-bp insert showed ten copies of a 48-bp repeat similar to D22Z3, with about 15% sequence deviation from the chromosome 22 consensus sequence. In situ hybridization of the newly isolated recombinant established its chromosome 21 specificity at high stringency. Physical mapping by pulsed field gel electrophoresis placed this new repeat in close vicinity to the chromosome 21 alphoid repeat. No cross-hybridization with other mammalian genomes except for those of apes was observed. The locus has been designated D21Z2 by the Genome Data Base. A gel mobility shift assay indicated that this repetitive motif has protein-binding properties.

Animals↗
PubMed1991

Identification and initial utilization of a portion of the smaller plasmid of Anabaena variabilis ATCC 29413 capable of replication in Anabaena sp. strain M-131.

Anabaena variabilis ATCC 29413 contains two cryptic plasmids. Clones of the smaller (41 kb) plasmid, designated pRDS1, in cosmid vectors were used to construct a physical map. A clone bank of pRDS1 constructed by ligating fragments from a XhoII digest of a pRDS1 cosmid clone into a mobilizable plasmid was used to locate an origin of replication of pRDS1. Because we were unable to cure A. variabilis of pRDS1, the clone bank was transferred by conjugation to another strain of Anabaena sp., strain M-131. A 5.3 kb fragment of pRDS1 contained all of the sequences necessary for replication in Anabaena sp. strain M-131 as judged by the ability to rescue the hybrid vector from exconjugants in unchanged from after many generations. Hybrid plasmids derived from pRDS1, one bearing genes for luciferase, were also transferred by conjugation to A. variabilis, where they appeared to recombine with pRDS1.

Chromosome Deletion↗
PubMed1989

Transformation by integration in Podospora anserina. III. Replacement of a chromosome segment by a two-step process.

We have developed in Podospora anserina a two-step procedure for DNA sequence replacement through transformation which might be applicable to other filamentous fungi. Targeting of transforming DNAs to their homologous locus is achieved provided a cosmid vector is used. Southern blot analysis of genomic DNAs from a set of transformants is presented. The data confirm that cosmids integrate into the chromosome through mostly homologous recombination which leads to a duplicated sequence separated by the vector. This event was found to be unstable in crosses. We show that this instability is due to the frequent excision of the vector together with the selective marker and one copy of the duplication, either the resident or foreign sequence. The two sequences can be distinguished because they exhibit restriction fragment length polymorphism. Therefore, Podospora anserina treats duplications occurring through transformation in a way differing from that exhibited by Neurospora crassa and Ascobolus immersus.

Ascomycota↗
PubMed1993

Molecular analysis of two genes between let-653 and let-56 in the unc-22(IV) region of Caenorhabditis elegans.

A previous study of genomic organization described the identification of nine potential coding regions in 150 kb of genomic DNA from the unc-22(IV) region of Caenorhabditis elegans. In this study, we focus on the genomic organization of a small interval of 0.1 map unit bordered on the right by unc-22 and on the left by the left-hand breakpoints of the deficiencies sDf9, sDf19 and sDf65. This small interval at present contains a single mutagenically defined locus, the essential gene let-56. The cosmid C11F2 has previously been used to rescue let-56. Therefore, at least some of C11F2 must reside in the interval. In this paper, we report the characterization of two coding elements that reside on C11F2. Analysis of nucleotide sequence data obtained from cDNAs and cosmid subclones revealed that one of the coding elements closely resembles aromatic amino acid decarboxylases from several species. The other of these coding elements was found to closely resemble a human growth factor activatable Na+/H+ antiporter. Paris of oligonucleotide primers, predicted from both coding elements, have been used in PCR experiments to position these coding elements between the left breakpoint of sDf19 and the left breakpoint of sDf65, between the essential genes let-653 and let-56.

Amino Acid Sequence↗
PubMed1992

Genetic analysis and complementation by germ-line transformation of lethal mutations in the unc-22 IV region of Caenorhabditis elegans.

The subject of this study is the organization of essential genes in the 2 map-unit unc-22 IV region of the Caenorhabditis elegans genome. With the goal of achieving mutational saturation of essential genes in this region, 6491 chromosomes mutagenized with ethyl methanesulfonate (EMS) were screened for the presence of lethal mutations in the unc-22 region. The genetic analysis of 21 lethal mutations in the unc-22 region resulted in the identification of 6 new essential genes, making a total of 36 characterized to date. A minimum of 49 essential genes are estimated to lie in this region. A set of seven formaldehyde-induced deficiencies of unc-22 and surrounding loci were isolated to facilitate the positioning of essential genes on the genetic and physical maps. In order to study essential genes at the molecular level, our approach was to rescue lethal mutations by the injection of genomic DNA in the form of cosmid clones into the germ-line of balanced heterozygotes carrying a lethal mutation. The cosmid clones containing let-56 and let-653 were identified by this method.

Animals↗
PubMed1990

Construction of an Azospirillum brasilense Sp7 recA mutant.

Cosmid clones encoding the recA gene of Azospirillum brasilense were isolated by intergeneric complementation of an Escherichia coli recA mutant. Site-directed Tn5 mutagenesis and subcloning of one complementing cosmid clone allowed us to localize the A. brasilense recA gene on a 1.2 kb DNA fragment. One Tn5 insertion that inactivates the cloned recA gene was crossed into the chromosome of A. brasilense by marker exchange. The resulting A. brasilense recA mutant showed increased sensitivity to the DNA methylating agent methyl methanesulfonate and to ultraviolet light and had at least one hundredfold reduced recombinational activity compared to the parent strain.

Azospirillum brasilense↗