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PubMed1994

Characterization of the murine beta-hexosaminidase (HEXB) gene.

The murine HEXB gene, encoding the beta-subunit of the lysosomal hydrolase, beta-hexosaminidase, was isolated from a mouse cosmid library as a single cosmid clone. The entire gene spans 22 kb, considerably less than the 40 kb spanned by its human counterpart. It is highly homologous to the human gene. The 14 intron-exon junctions are entirely conserved, although the intron sequences diverge rapidly. Upstream of the coding region, a 1.3 kb segment was sequenced and shown to function as a promoter when fused with a reporter gene and expressed in monkey COS-7 cells. A short sequence (100 bp), near the start of the coding region, exhibits strong homology to the human HEXB promoter. Analysis of the tissue distribution of the HEXB mRNA in 129/Sv male mice revealed up to 28-fold tissue-specific variations in transcript levels. The kidney and the epididymis had the highest mRNA levels consistent with past surveys of enzyme activity.

Amino Acid Sequence↗
PubMed2001

Characterisation of three chromosomal ends of Leishmania major reveals transcriptional activity across arrays of reiterated and unique sequences.

The 36 chromosomes of the parasite Leishmania major range in size from 200 kb to approximately 2.5 Mb and variation between homologues seems to be restricted to the telomeric and subtelomeric regions. We have isolated three cosmids carrying the telomere hexameric repeat and assigned them to the extreme location of chromosomes 3, 7 and 20. When considering the distribution of repetitive sequences, Southern analysis of the three chromosomal ends indicated the existence of at least two classes of chromosomal extremities: one of them is composed almost exclusively of unique sequences and the other is characterised by patches of both reiterated and unique sequences. We devised a transfection-based strategy that allowed the determination of a map of transcripts in each of the regions examined. Sequencing of the chromosome 20 cosmid revealed the existence of a novel class of reiterated sequence, LST-R378, and 10 ORFs drawing a map of putative genes compatible with the map of transcripts.

Animals↗
PubMed1998

The phenotype enhancement method identifies the Xcp outer membrane secretion machinery from Pseudomonas alcaligenes as a bottleneck for lipase production.

Pseudomonas alcaligenes M-1 has been selected from an intensive screening for micro-organisms that can naturally produce a lipase active in detergent formulations. The lipase expression has been increased to allow high level secretion from Pseudomonas alcaligenes, via the introduction of multi-copy plasmids. In order to improve the lipase yield further, the phenotype enhancement method has been developed. This idea comprises the reintroduction of a cosmid library with random chromosomal fragments in a P. alcaligenes strain with already high lipase productivity. One of the strains which showed an enhanced lipase production appeared to contain a cosmid encoding the outer membrane secretion genes. These xcp-genes are clustered in two divergently transcribed operons similar to the situation in Pseudomonas aeruginosa. Remarkably and dissimilar to P. aeruginosa, in between the two xcp gene clusters, two reading frames of unknown function--OrfV and OrfX--are present. For OrfX no equivalent can be found in the known protein data bases. On the other hand, OrfV shows homology to the regulatory proteins MalT and AcoK. Some evidence is provided that suggests that OrfV acts as a regulator of the xcp operons. A model is proposed for the regulation of the secretion system from P. alcaligenes.

Amino Acid Sequence↗
PubMed1999

Partial mapping and sequencing of a fish iridovirus genome reveals genes homologous to the frog virus 3 p31, p40 and human eIF2alpha.

Iridovirus-like pathogens have been recognized as a cause of serious systemic diseases among feral, cultured and ornamental fish in the recent years. Mortalities of fish due to systemic iridovirus infection reaching 30-100% were observed in Europe, Australia, Japan and Thailand. Up to now, the molecular biology of these important pathogens has been poorly documented. To get better insights on the genomic organization of these piscine iridoviruses, we have constructed a cosmid viral DNA library from the epizootic hematopoietic necrosis virus (EHNV). Two recombinant cosmids (Cos7 and Cos12) have been selected for systematic sequencing. Cos7 and 12 are localized side by side along the genome and cover the 2/3 part of the total EHNV genome which has been estimated to be approximately 101.47 kb in length. Thirty five kilobase pairs (kbps) from Cos7 and 10 kbps from Cos12 have been determined. Sequence analysis revealed open reading frames (ORF) sharing homologies with sequences from the Frog virus 3 such as the p31 and p40 proteins. Among the others identified ORFs, some of them presented homologies with known protein sequences, such as the human eIF2alpha protein, and some did not show any significant homologies with sequences available in the databases. But, none were related to Lymphocystis virus, a member of the Iridoviridae family, for which the full genome nucleotide sequence has been determined.

Amino Acid Sequence↗
PubMed1997

Use of ordered deletions in genome sequencing.

Previous attempts to use the non-random approach for sequencing long DNA fragments have met with little success. As a result, nearly all genomic sequencing is done by the random (shotgun) approach, and the economy promised by the non-random approach has so far not materialized. Here we describe a simple system based on the use of ordered deletions that can be incorporated in the common strategies for genome sequencing. Long genomic fragments are cloned in the pAL-F cosmid and fragmented by digestion with specific restriction endonucleases. The digests are religated to subclone individual restriction fragments. The subclones are then subdivided by overlapping deletions and used for sequencing. We present the nucleotide sequences of two cosmid inserts from chromosome IV of Drosophila (containing the ci gene and the 5' end of the zfh-2 gene) that were determined by this method. This is the first report of successful sequencing of long genomic fragments by the use of overlapping deletions. Our calculations show that, with the present approach, sequence data can be acquired at a rate comparable to the shotgun approach but with significantly reduced numbers (approximately 30%) of sequencing runs. Hence, the use of ordered deletions should allow significant savings in both the amount and cost of sequencing work.

Animals↗
PubMed1997

Molecular characterization of a serine/threonine kinase in the DiGeorge minimal critical region.

The majority of patients with DiGeorge, velocardiofacial or conotruncal anomaly facial syndromes share a common genetic etiology, deletion of chromosomal region 22q11.2. This report describes a computational approach toward the identification and molecular characterization of a newly identified serine/threonine kinase from the minimal critical deleted region (MDGCR). A cosmid contig of the minimal critical region has been assembled and sequenced in its entirety. Database searches and computer analysis of one cosmid (111f11) for coding sequences identified two regions with high similarity to the mouse serine/threonine kinase, Tsk1. Our investigations demonstrate that one of these regions contains a testis-specific gene that undergoes differential splicing, while the other region is most likely a pseudogene. Northern blot analysis and cDNA cloning demonstrate that there is alternate processing of the 3'UTR without altering the conserved kinase domains within the open reading frame. Serine/threonine kinases can play a regulatory role and have been found to be expressed during early embryogenesis. Based on its position in the MDGCR and possible function, the gene reported here is a candidate for the features seen in the 22q11 deletion syndrome.

Alternative Splicing↗
PubMed1997

Positioning of 72 potentially full size LTRs of human endogenous retroviruses HERV-K on the human chromosome 19 map. Occurrences of the LTRs in human gene sites.

Seventy-two near full size long terminal repeats (LTRs) of human endogenous retrovirus of K-family (HERV-K) have been precisely located on the metric map of human chromosome 19. The LTR-related sequences were identified and assigned to cosmids by hybridization with two independent chromosome 19 specific cDNA clones corresponding to different parts of U3 region of LTR of HERV-K. The presence of full-size LTR sequences in a cosmid was further verified by PCR assay with a pair of primers complementary to the termini of the LTR. Coincidences of the LTR and the known genes positions are discussed.

Base Sequence↗
PubMed1997

Cloning and sequencing of complement component C9 and its linkage to DOC-2 in the pufferfish Fugu rubripes.

The Japanese pufferfish Fugu rubripes has a 400 Mb genome with high gene density and minimal non-coding complexity, and is therefore an ideal vertebrate model for sequence comparison. The identification of regions of conserved synteny between Fugu and humans would greatly accelerate the mapping and ordering of genes. Fugu C9 was cloned and sequenced as a first step in an attempt to characterize the region in Fugu homologous to human chromosome 5p13. The 11 exons of the Fugu C9 gene share 33% identity with human C9 and span 2.9 kb of genomic DNA. By comparison, human C9 spans 90 kb, representing a 30-fold difference in size. We have also determined by cosmid sequence scanning that DOC-2, a tumour suppresser gene which also maps to human 5p13, lies 6-7 kb from C9 in a head-to-head or 5' to 5' orientation. These results demonstrate that the Fugu C9/DOC-2 locus is a region of conserved synteny. Sequence scanning of overlapping cosmids has identified two other genes, GAS-1 and FBP, both of which map to human chromosome 9q22, and lie adjacent to the Fugu C9/DOC-2 locus, indicating the boundary between two syntenic regions.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Detection of type III secretion system genes in animal isolates of Bordetella bronchiseptica.

A cosmid clone bank of Bordetella bronchiseptica genomic DNA was screened for the presence of type III secretion (TTS) genes using a probe derived from the TTS system genes of Ralstonia solanacearum. A 3.35kb PstI fragment, sub-cloned from a hybridising cosmid clone, was sequenced and found to contain a 97bp overlap with the previously reported B. bronchiseptica bscIJKLNO TTS gene cluster. DNA and predicted protein homology analysis suggests that a bscPQRST cluster lies immediately downstream of bscIJKLNO. A PCR amplification assay indicated that the bscT locus was present in 27 B. bronchiseptica animal isolates tested (100%). Dot-blot DNA hybridisation using probes for bscT and bscP confirmed the presence of these loci in six canine isolates associated with a variety of clinical signs. Although TTS has been implicated in the pathogenicity of B. bronchiseptica, it is likely that different clinical manifestations may be due to variations in gene expression or host factors, rather than the absence or presence of TTS genes.

Amino Acid Sequence↗
PubMed1992

Detailed mapping around the breakpoint of (3;8) translocation in familial renal cell carcinoma and FRA3B.

As a critical step toward cloning the breakpoint of the (3;8) translocation observed in familial renal cell carcinoma and a common fragile site on chromosome 3p (FRA3B), we have characterized the 3p14 region containing the breakpoint and FRA3B by fluorescence in situ hybridization (FISH), pulsed-field gel electrophoresis (PFGE), and genetic linkage analysis. Of 23 cosmids mapped by FISH, 14 cosmids were distal to the breakpoint of t(3;8) and 9 were proximal. Analyses of FRA3B by FISH were identical to those for the (3;8) breakpoint and indicated that the breakpoint of t(3;8) occurred at or very close to the fragile site. We have also constructed a genetic linkage map and a preliminary long-range restriction map using PFGE of the 3p14 region. The linkage results were consistent with the physical data. The combined results of FISH, PFGE, and linkage analysis establish a basis for further experiments to clone the (3;8) breakpoint and FRA3B.

Animals↗
PubMed1998

Replication of the linear chromosomal DNA from the centrally located oriC of Streptomyces ambofaciens revealed by PFGE gene dosage analysis.

From a cosmid clone of Streptomyces ambofaciens containing the dnaA and gyrAB genes, a 2.7-kb self-replicating DNA fragment containing the chromosome replication origin oriC was isolated. This cosmid was previously maped physically to a region near the middle of the 8-Mb linear chromosomal DNA. A pulsed-field gel electrophoresis time-course analysis revealed that sequences flanking oriC were overrepresented relative to the rest of the chromosomal DNA during rapid growth, indicating that this origin is active. In addition, the terminal regions of the chromosomal DNA showed a slight overrepresentation at the onset of stationary phase.

Amino Acid Sequence↗
PubMed1996

The mouse col11a2 gene. Some transcripts from the adjacent rxr-beta gene extend into the col11a2 gene.

Type XI collagen is present in small amounts in cartilage, together with small amounts of type IX and type V collagens and large amounts of type II collagen. Here, primers based on the nucleotide sequences of partial human cDNAs and mouse genomic DNAs that were analyzed by other investigators were used to isolate a cDNA for the mouse col11a2 gene. Cosmid clones for the mouse col11a2 gene were isolated, and 12.4 kb of the nucleotide sequences were defined. Analysis of the genomic sequences identified three exons in the mouse gene that were recently shown to undergo alternative splicing (Tsumaki and Kimura, J. Biol. Chem. 270, 2372-2378, 1995; Zhidkova et al., J. Biol. Chem. 270, 94886-9493, 1995). In addition, analysis of the cosmid clones revealed that the 5' end of the mouse col11a2 gene was located head-to-tail with the mouse retinoic X receptor beta gene. RT-PCR assays demonstrated that some transcripts from the retinoic X receptor beta gene extend into the col11a2 gene. Therefore, there may be coordinate expression of the two genes.

Animals↗
PubMed1997

Structural mapping of the dihydrofolate reductase amplicon in mosquito cells resistant to methotrexate.

A cosmid library containing genomic DNA from mosquito cells in which the dihydrofolate reductase gene had become amplified in response to methotrexate selection was used to define the structure of the amplified DNA region (amplicon). A series of overlapping cosmids identified more than 200 kb of amplicon DNA, which was mapped relative to BssHII fragments from genomic DNA separated by transverse alternating field electrophoresis. These analyses indicated that, in Mtx-5011-256 mosquito cells, dhfr genes occur in at least two types of amplicon. The predominant Type I amplicon measures at least 215 kb and contains most of the dhfr genes. In addition, a small proportion of dhfr genes reside in a Type II amplicon, which is arranged in head-to-tail tandem repeats measuring 162 kb. Both amplicons share at least 70 kb of DNA sequence at their 5' ends. Approximately 20 fragments containing repeated sequence elements have been identified in the cloned amplicon DNA. Hybridization of amplicon DNA fragments containing repeated sequences to genomic DNA detected polymorphisms between wild type and methotrexate-resistant cells, suggesting that recombination may generate the divergence observed at the 3'-ends of Type I and Type II amplicons. This first detailed analysis of an insect dhfr amplicon provides an essential basis for ongoing investigation of repeated sequences, transcribed units and replication origins within the amplicon DNA.

Aedes↗
PubMed1998

The granaticin biosynthetic gene cluster of Streptomyces violaceoruber Tü22: sequence analysis and expression in a heterologous host.

BACKGROUND: The granaticins are members of the benzoisochromanequinone class of aromatic polyketides, the best known member of which is actinorhodin made by Streptomyces coelicolor A3(2). Genetic analysis of this class of compounds has played a major role in the development of hypotheses about the way in which aromatic polyketide synthases (PKSs) control product structure. Although the granaticin nascent polyketide is identical to that of actinorhodin, post-PKS steps involve different pyran-ring stereochemistry and glycosylation. Comparison of the complete gene clusters for the two metabolites is therefore of great interest. RESULTS: The entire granaticin gene cluster (the gra cluster) from Streptomyces violaceoruber T-22 was cloned on either of two overlapping cosmids and expressed in the heterologous host, Streptomyces coelicolor A3(2), strain CH999. Chemical analysis of the recombinant strains demonstrated production of granaticin, granaticin B, dihydrogranaticin and dihydrogranaticin B, which are the four known metabolites of S. violaceoruber. Analysis of the complete 39,250 base pair sequence of the insert of one of the cosmids, pOJ466-22-24, revealed 37 complete open reading frames (ORFs), 15 of which resemble ORFs from the act (actinorhodin) gene cluster of S. coelicolor A3(2). Among the rest, nine resemble ORFs potentially involved in deoxysugar metabolism from Streptomyces spp. and other bacteria, and six resemble regulatory ORFs. CONCLUSIONS: On the basis of these resemblances, putative functional assignments of the products of most of the newly discovered ORFs were made, including those of genes involved in the PKS and tailoring steps in the biosynthesis of the granaticin aglycone, steps in the deoxy sugar pathway, and putative regulatory and export functions.

Amino Acid Sequence↗
PubMed2001

Genomic analysis of MIC genes in rhesus macaques.

MIC genes map to the major histocompatibility complex (MHC) and are distantly related to MHC class I genes. Recently, MICA/MICB-like genes have been described in nonhuman primates. In Macaca mulatta, three MICA/B-like genes could be identified: Mamu-MIC1, Mamu-MIC2, and Mamu-MIC3. We show here the isolation and characterization of rhesus macaque cosmid clones which carry the Mamu-MIC2 and Mamu-MIC3 genes. Neither the MIC2- and MIC3-coding sequences nor respective flanking sequences can be aligned unambiguously to either the human HLA-MICA or -MICB subregions, although MIC2 was found at a similar distance to the BAT1 gene as known for MICB in human. Thus, the characteristics allowing for a classification of primate MIC genes as being of the MICA or MICB types appear to have evolved after the separation of humans and rhesus monkeys from a common ancestor. Furthermore, also Mamu-MICD-containing cosmids could be isolated. In contrast to Mamu-MIC2 and Mamu-MIC3, the Mamu-MICD gene and its flanking sequences are highly conserved and orthologous to the human MICD subregion.

Animals↗
PubMed2000

Identification of a Salmonella typhimurium genomic region involved in invasion of HeLa and Henle-407 epithelial cells.

To identify the invasion determinant, a cosmid library was constructed by cloning a genomic library of Salmonella typhimurium 82/6915 into a cosmid vector, pLA2917. A genomic region involved in invasion of cultured HeLa and Henle-407 cells was subcloned into plasmid pGEM-7Z. E. coli strain DH1 carrying pSV6235 consisting of a S. typhimurium 4.6 kb genomic region in pGEM-7Z showed invasion of cultured HeLa and Henle-407 cells. Nested sequential deletions were introduced into the 4.6 kb genomic region of pSV6235. The E. coli recombinants which contained less than 1.5 kb deletions from the 5' end (SmaI site) of the genomic region invaded the cells as effectively as DH1 (pSV6235). The invasion of the recombinants carrying over 2.0 kb deletions from the end of pSV6235 was significantly inactivated compared to DH1 (pSV6235). Restriction enzyme analysis showed that the 3.1 kb fragment from the 3' end of the 4.6 kb genomic region was distinguished from the Salmonella pathogenicity I genes of S. typhimurium such as the inv, spa, and hil regions showing invasion of the cultured eukaryotic cells.

Cell Line↗
PubMedDate not supplied

Isolation, sequence determination and expression in Escherichia coli of the isopenicillin N synthetase gene from Cephalosporium acremonium.

The enzyme isopenicillin N synthetase (IPS) catalyses the oxidative condensation of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (LLD-ACV) to isopenicillin N, which is a central reaction in the pathway to clinically important penicillins and cephalosporins. Here we report the cloning, characterization and expression in Escherichia coli of the gene encoding the IPS protein in Cephalosporium acremonium. The IPS gene was identified by purifying IPS protein, determining the first 23 amino-terminal amino acids, preparing a set of synthetic oligonucleotides encoding a portion of the determined amino-acid sequence, and probing a cosmid genome library with the mixed oligonucleotides. A cosmid hybridizing with the probe was isolated and the IPS gene was localized and sequenced. The IPS gene encodes a polypeptide of relative molecular mass (Mr) 38,416. When this open reading frame was cloned into an E. coli expression vector and inserted into E. coli, the recombinant E. coli produced a new protein co-migrating with authentic IPS as the major protein of the cell (approximately 20% of cell protein). Crude cell extracts condensed LLD-ACV to a penicillinase-sensitive molecule whose antibacterial activity indicated that it was isopenicillin N.

Acremonium↗
PubMed1998

The tomato Mi-1 gene confers resistance to both root-knot nematodes and potato aphids.

Mi-1, a Lycopersicon peruvianum gene conferring resistance to the agricultural pests, root-knot nematodes, and introgressed into tomato, has been cloned using a selective restriction fragment amplification based strategy. Complementation analysis of a susceptible tomato line with a 100 kb cosmid array yielded a single cosmid clone capable of conferring resistance both to the root-knot nematode Meloidogyne incognita and to an unrelated pathogen, the potato aphid Macrosiphum euphorbiae. This resistance was stable. The Mi-1 gene encodes a protein sharing structural features with the nucleotide-binding site leucine-rich repeat-containing type of plant resistance genes.

Amino Acid Sequence↗