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PubMed1998

Characterization of the human neurocan gene, CSPG3.

Neurocan is a chondroitin sulfate proteoglycan thought to be involved in the modulation of cell adhesion and migration. Its sequence has been determined previously in rat and mouse (Rauch et al., 1992. Cloning and primary structure of neurocan, a developmentally regulated, aggregating, chondroitin sulfate proteoglycan of the brain. J. Biol. Chem. 267, 19536-19547; Rauch et al., 1995. Structure and chromosomal location of the mouse neurocan gene. Genomics 28, 405-410). We describe here the complete coding sequence of the human neurocan mRNA, known as CSPG3, as well as mapping data, expression analysis, and genomic structure. A cDNA known as CP-1 was initially sequenced as part of a gene discovery project focused on characterizing chromosome 19-specific cDNAs. Sequence homology searches indicated close homology to the mouse and rat proteoglycan, neurocan (GenBank accession Nos X84727 and M97161). Northern analysis identified a brain-specific transcript of approx. 7.5kb. A longer cDNA clone, GT-5, was obtained, fine-mapped to the physical map of chromosome 19 by hybridization to a chromosome-specific cosmid library, and sequenced. Full coding sequence of the mRNA indicates a 3963bp open reading frame corresponding to a 1321 amino acid protein, similar to the protein length found in mouse and rat. The amino acid sequence of human neurocan shows 63% identity with both the mouse and rat sequences. Finally, genomic sequencing of a cosmid containing the complete neurocan gene was performed to determine the genomic structure of the gene, which spans approx. 41kb, and is transcribed in the telomere to centromere orientation.

Amino Acid Sequence↗
PubMed1999

Structures, sequence characteristics, and synteny relationships of the transcription factor E4TF1, the splicing factor U2AF35 and the cystathionine beta synthetase genes from Fugu rubripes.

A cosmid containing the beta-amyloid precursor protein (APP) from Fugu rubripes has been completely sequenced. In addition to APP, the cosmid contains the E4TF1-60 transcription factor, the U2AF35 pre-mRNA splicing factor, and the cystathionine beta synthetase (CBS) gene. The human homologues of all four genes map to human chromosome 21 but are not clustered; APP and E4TF1-60 map within 21q21, whereas U2AF35 and CBS map approximately 20Mb distal in 21q22. 3. The protein sequences of the Fugu genes vary in their overall level of similarity to their mammalian homologues, but several regions of functional importance are almost identical. As expected, the intron/exon structures of the homologous pairs of genes are highly conserved, but there are significant differences in the compaction ratios. The introns of APP and E4TF1-60 are 49- and 24-fold smaller in Fugu than in human, and the intergenic distance is compressed at least 100-fold. For U2AF35 and CBS, the introns are compressed only five- to eightfold. These size differences were compared with those for a number of previously reported Fugu genes; in general, levels of compaction of Fugu genes are consistent with the isochore locations of the human homologues.

Amino Acid Sequence↗
PubMed1999

Fine mapping of a polymorphic CA repeat marker on human chromosome 19 and its use in population studies.

A highly polymorphic microsatellite (CA)n-marker (CAct685) previously isolated from human chromosome 19 cosmid library was localized near GPI in 19q13.1. For the fine localization of this marker, the hybridization with chromosome 19-specific cosmid libraries assembled in contigs was used. Polymorphism analysis of the marker in 12 populations of Russia and neighboring countries showed 14 alleles containing from 16 to 30 repeat units. Populations belonging to Indo-European, Uralic and Altaic linguistic families demonstrated a great similarity in allele frequency profiles. Differences between these populations were lower for CAct685 than for classical markers. Allele distribution of CAct685 in a Chukchi population belonging to the Chukchi-Kamchatkan linguistic family differs from those in all other populations, that may be typical for Mongoloid population or reflect an ethnic history of Chukchi as a small population. Thus use of the CAct685 marker seems to be effective for analysis of distant peoples.

Alleles↗
PubMed1992

Structure and linkage of the D2 dopamine receptor and neural cell adhesion molecule genes on human chromosome 11q23.

The gene encoding the D2 dopamine receptor (DRD2) is located on human chromosome 11q23 and has been circumstantially associated with a number of human disorders including Parkinson's disease, schizophrenia, and susceptibility to alcoholism. To determine the physical structure of the DRD2 gene, we utilized cosmid cloning, isolation of yeast artificial chromosomes (YACs), and pulsed-field gel electrophoresis to construct a long-range physical map of human chromosome 11q23 linking the genes for the DRD2 and neural cell adhesion molecule (NCAM). The D2 dopamine receptor gene extends over 270 kb and includes an intron of approximately 250 kb separating the putative first exon from the exons encoding the receptor protein. The resulting physical map spans more than 1.5 mb of chromosome band 11q23 and links the DRD2 gene with the gene encoding the NCAM located 150 kb 3' of the DRD2 gene and transcribed from the same DNA strand. We additionally located the sites of at least four hypomethylated HTF islands within the physical map, which potentially indicate the sites of additional genes. High-resolution fluorescent in situ suppression hybridization using cosmid and YAC clones localized this gene cluster between the ApoAI and STMY loci at the interface of bands 11q22.3 and 11q23.1.

Base Sequence↗
PubMed1992

Arrangement and localization of the human GM-CSF receptor alpha chain gene CSF2RA within the X-Y pseudoautosomal region.

The gene encoding one subunit of the receptor for the hemopoietic growth factor, GM-CSF, has been previously localized to the short arm of the human sex chromosomes. By pulsed-field gel electrophoresis, the precise localization of this gene, CSF2RA, within the pseudoautosomal region has been determined. The gene is located 1180 to 1300 kb from the telomere, in close proximity to the CpG island B5. The CSF2RA gene spans at least 45 kb, and a representation of most of the gene on three overlapping cosmid clones has been obtained. The exon(s) encoding the first 35 bp of cDNA sequence lies outside these cosmids. The CSF2RA gene is characterized by abundant hypervariable sequences, and a number of informative restriction fragment length polymorphisms have been defined.

Amino Acid Sequence↗
PubMed1998

Clinical diagnosis of heterozygous dystrophin gene deletions by fluorescence in situ hybridization.

Two-thirds of patients affected by Duchenne or Becker muscular dystrophy (DMD/BMD) carry large intra-genic deletions in the dystrophin gene. In males, the deletions can be efficiently detected using multiplex polymerase chain reaction (PCR) and Southern blotting. In contrast, deletion detection in carrier females is complicated by the presence of a normal gene copy on the second X-chromosome. We have analyzed the boundaries of 570 deletions and 34 duplications in the dystrophin gene identified in the São Paulo and Leiden diagnostic laboratories. The data were used to select an optimal set of cosmid probes for the detection of the most frequently deleted areas of the dystrophin gene. Six cosmids were evaluated in fluorescence in situ hybridization (FISH) experiments to assess deletions in 21 heterozygous deletion-carriers and nine controls. No discrepancy was found between the FISH analysis and the molecular data, demonstrating the accuracy of the technique for carrier detection in Duchenne and Becker muscular dystrophy.

DNA Probes↗
PubMed1997

Immunological screening of a genomic M. bovis BCG library expressed in M. smegmatis and identification of the M. bovis BCG analog of ClpB.

This work describes the screening of a M. bovis BCG cosmid library in M. smegmatis with a hyperimmune rabbit anti-BCG serum. Cross-reactive antibodies interfere with the detection of BCG specific antigens in M. smegmatis culture filtrates. We, therefore, screened parallel western blots with serum adsorbed with a M. smegmatis cell lysate and unadsorbed serum. Comparison of the western blots allowed distinction between BCG specific and cross-reactive M. smegmatis antigens. Thirty-one cosmids expressed BCG specific antigens. One of them, a hitherto undescribed 100 kDa antigen was subcloned, sequenced and expressed in E. coli. It shows a high degree of homology to ClpB, a member of the Clp family of proteases and was immunologically reactive with the rabbit hyperimmune serum against M. bovis BCG. A positive signal was also obtained with sera of patients with tuberculosis. This antigen is a previously unrecognized target of the human immune response to mycobacteria.

Animals↗
PubMed2001

Identification of a sugar flexible glycosyltransferase from Streptomyces olivaceus, the producer of the antitumor polyketide elloramycin.

BACKGROUND: Elloramycin is an anthracycline-like antitumor drug related to tetracenomycin C which is produced by Streptomyces olivaceus Tü2353. Structurally is a tetracyclic aromatic polyketide derived from the condensation of 10 acetate units. Its chromophoric aglycon is glycosylated with a permethylated L-rhamnose moiety at the C-8 hydroxy group. Only limited information is available about the genes involved in the biosynthesis of elloramycin. From a library of chromosomal DNA from S. olivaceus, a cosmid (16F4) was isolated that contains part of the elloramycin gene cluster and when expressed in Streptomyces lividans resulted in the production of a non-glycosylated intermediate in elloramycin biosynthesis, 8-demethyl-tetracenomycin C (8-DMTC). RESULTS: The expression of cosmid 16F4 in several producers of glycosylated antibiotics has been shown to produce tetracenomycin derivatives containing different 6-deoxysugars. Different experimental approaches showed that the glycosyltransferase gene involved in these glycosylation events was located in 16F4. Using degenerated oligoprimers derived from conserved amino acid sequences in glycosyltransferases, the gene encoding this sugar flexible glycosyltransferase (elmGT) has been identified. After expression of elmGT in Streptomyces albus under the control of the erythromycin resistance promoter, ermEp, it was shown that elmG can transfer different monosaccharides (both L- and D-sugars) and a disaccharide to 8-DMTC. Formation of a diolivosyl derivative in the mithramycin producer Streptomyces argillaceus was found to require the cooperative action of two mithramycin glycosyltransferases (MtmGI and MtmGII) responsible for the formation of the diolivosyl disaccharide, which is then transferred by ElmGT to 8-DMTC. CONCLUSIONS: The ElmGT glycosyltransferase from S. olivaceus Tü2353 can transfer different sugars into the aglycon 8-DMTC. In addition to its natural sugar substrate L-rhamnose, ElmGT can transfer several L- and D-sugars and also a diolivosyl disaccharide into the aglycon 8-DMTC. ElmGT is an example of sugar flexible glycosyltransferase and can represent an important tool for combinatorial biosynthesis.

Anthraquinones↗
PubMed1996

Distribution, gene sequence and expression in vivo of the plasmid encoded fimbrial antigen of Salmonella serotype Enteritidis.

The pefA gene which encoded the serotype associated plasmid (SAP) mediated fimbrial major subunit antigen of Salmonella enterica serotype Typhimurium shared genetic identity with 128 of 706 salmonella isolates as demonstrated by dot (colony) hybridization. Seventy-seven of 113 isolates of Typhimurium and individual isolates of serotypes Bovis-morbificans, Cholerae-suis and Enteritidis phage type 9b hybridized pefA strongly, whereas 48 isolates of Enteritidis hybridized pefA weakly and one Enteritidis isolate of phage type 14b failed to hybridize. Individual isolates of 294 serotypes and 247 individual isolates of serotype Dublin did not hybridize pefA. Southern hybridization of plasmids extracted from Enteritidis demonstrated that the pefA gene probe hybridized strongly an atypical SAP of 80 kb in size harboured by one Enteritidis isolate of phage-type 9b, whereas the typical SAP of 58 kb in size harboured by 48 Enteritidis isolates hybridized weakly. One Enteritidis isolate of phage type 14b which failed to hybridize pefA in dot (colony) hybridization experiments was demonstrated to be plasmid free. A cosmid library of Enteritidis phage type 4 expressed in Escherichia coli K12 was screened by hybridization for the presence of pef sequences. Recombinant clones which were deduced to harbour the entire pef operon elaborated a PEF-like fimbrial structure at the cell surface. The PEF-like fimbrial antigen was purified from one cosmid clone and used in western blot experiments with sera from chickens infected with Enteritidis phage-type 4. Seroconversion to the fimbrial antigen was observed which indicated that the Enteritidis PEF-like fimbrial structure was expressed at some stage during infection. Nucleotide sequence analysis demonstrated that the pefA alleles of Typhimurium and Enteritidis phage-type 4 shared 76% DNA nucleotide and 82% deduced amino acid sequence identity.

Adhesins, Bacterial↗
PubMedDate not supplied

Molecular cloning of the gamma-glutamyltranspeptidase gene from a Pseudomonas strain.

gamma-Glutamyltranspeptidase (GGT) was purified from a Pseudomonas sp. strain A14. The purified enzyme was found to be composed of two nonidentical subunits with molecular weights of 39,000 and 22,000 and had a pI of > 8.6. The partial N-terminal amino acid sequences of both subunits and some proteolytic fragments were determined. Using mixed oligonucleotides designed from the partial amino acid sequences as hybridization probes, one cosmid clone which contained the GGT gene was isolated from a Pseudomonas sp. strain A14 cosmid genome library, and the DNA sequence of the GGT gene was determined. The nucleotide sequence and the protein sequence analysis revealed that GGT was synthesized as a precursor protein of 575 amino acids and then processed to mature enzyme, presumably after removal of a signal peptide. Comparison of the predicted amino acid sequence of Pseudomonas GGT with published results for Escherichia coli K-12 and rat kidney GGTs shows that the protein sequence of Pseudomonas GGT is 51% and 33% identical to the E. coli and rat GGT sequences, respectively. Higher similarity is observed among the small subunits, which have been thought to have a binding site for the gamma-glutamyl residue. Expression of the cloned Pseudomonas GGT gene in E. coli was subjected to Western blot analysis using antibody raised against the purified GGT. This suggested that processing of the precursor protein to its subunits is temperature-dependent, because the amount of mature GGT protein was increased when the culture was performed at low temperature.

Amino Acid Sequence↗
PubMed2001

The Rhodobacter capsulatus genome.

The genome of Rhodobacter capsulatus has been completely sequenced. It consists of a single chromosome containing 3.5 Mb and a circular plasmid of 134 kb. This effort, started in 1992, began with a fine-structure restriction map of an overlapping set of cosmids that covered the genome. Cosmid sequencing led to a gapped genome that was filled by primer walking on the chromosome and by using lambda clones. Methods had to be developed to handle strong stops in the high GC (68%) inserts. Annotation was done with the ERGO system at Integrated Genomics, as was the reconstruction of the cell's metabolism. It was possible to recognize 3709 orfs of which functional assignments could be made with high confidence to 2392 (65%). Unusual features include the presence of numerous cryptic phage genomes embedded in the chromosome.

Journal Article↗
PubMed2001

Effective generation of very low density lipoprotein receptor transgenic mice by overlapping genomic DNA fragments: high testis expression and disturbed spermatogenesis.

The generation of functional transgenes via microinjection of overlapping DNA fragments has previously been reported to be successful, but it is still not a widely applied approach. Here we show that the method is very reliable, and should be considered, in case a single large insert clone of the desired gene is not available. In the present study, two large DNA fragments consisting of overlapping cosmids, together constituting the human very low density lipoprotein receptor (VLDLR) gene (35 kb), were used to generate VLDLR transgenic (VLDLR-Tg) mice. Three transgenic founders were born, of which two (strain #2 and #3) generated transgenic offspring. Using Fiber-FISH analysis, the integration site was shown to contain at least 44 and 64 DNA fragments in mouse strains #2 and #3, respectively. This copy number resulted in integration sites of 1.5 and 2.5 megabase in size. Notably, over 90% of the fragments in both mouse strains #2 and #3 were flanked by their complementary fragment. In line with this observation, Southern blot analysis demonstrated that the correct recombination between fragments predominated in the transgenic insertion. Human VLDLR expression was detected in testis, kidney and brain of both mouse strains. Since this pattern did not parallel the endogenous VLDLR expression, some crucial regulatory elements were probably not present in the cosmid clones. Human VLDLR expression in testis was detected in germ cells up to the meiotic stage by in situ mRNA analysis. Remarkably, in the F1 generation of both VLDLR-Tg mouse strains the testis was atrophic and giant cells were detected in the semineferous tubuli. Furthermore, male VLDLR-Tg mice transmitted the transgene to their progeny with low frequencies. This could imply that VLDLR overexpression in the germ cells disturbed spermatogenesis.

Alternative Splicing↗
PubMed1982

Arrangement of human immunoglobulin heavy chain constant region genes implies evolutionary duplication of a segment containing gamma, epsilon and alpha genes.

Cosmid clones containing the human gamma, epsilon and alpha heavy chain constant region genes and an epsilon pseudogene have been isolated. All these genes have a switch sequence detectable by hybridization. We have studied overlapping cosmids covering two separate regions of the genome, and the gene order in each of these regions was found to be gamma-gamma-epsilon-alpha. This implies an evolutionary duplication in this multigene family involving gamma, epsilon and alpha genes.

Biological Evolution↗
PubMedDate not supplied

Pseudoautosomal DNA sequences in the pairing region of the human sex chromosomes.

A DNA probe from a human Y chromosome-derived cosmid detects a single-copy genomic DNA fragment which can appear in different allelic forms shared by both sex chromosomes. Variants at this DNA locus show an autosomal pattern of inheritance, undergo recombination with sexual phenotype and can therefore be described as 'pseudoautosomal'. Another probe from the same cosmid detects a sequence repeated 15-20 times per haploid genome. These repeats also appear pseudoautosomal and map exclusively to the short-arm terminal region of each sex chromosome.

Chromosome Mapping↗
PubMed2005

Combination of WAGR and Potocki-Shaffer contiguous deletion syndromes in a patient with an 11p11.2-p14 deletion.

Aniridia, Wilms tumor, genitourinary abnormalities, growth and mental retardation are the cardinal features of the WAGR 11p13 deletion syndrome. The Potocki-Schaffer syndrome or proximal 11p deletion syndrome (previously DEFECT11 syndrome) is a contiguous gene syndrome associated with deletions in 11p11.2, principal features of which are multiple exostoses and enlarged parietal foramina. Mental handicap, facial dysmorphism and craniosynostosis may also be associated. We report a patient with combined WAGR and Potocki-Shaffer syndromes, and obesity. She presented with aniridia, cataract, nystagmus, corneal ulcers and bilateral congenital ptosis. A left nephroblastoma was detected at 15 months. Other features included moderate developmental delay, growth deficiency, facial dysmorphism, multiple exostoses and cranial lacunae. High-resolution and molecular cytogenetics confirmed a del(11)(p11.2p14.1) deletion with a proximal breakpoint between the cosmid DO8153 and the BAC RP11-104M24 to a distal breakpoint between cosmids CO8160 (D11S151) and F1238 (D11S1446). The deletion therefore includes EXT2, ALX4, WT1 and PAX6. This case appears to be the second patient reported with this combined deletion syndrome and confirms the association of obesity in the WAGR spectrum, a feature previously reported in four cases, and for which the acronym WAGRO has been suggested. Molecular and follow-up data on the original WAGRO case are briefly presented.

Adolescent↗
PubMed2000

New tools for the generation of E1- and/or E3-substituted adenoviral vectors.

We have designed new vectors for the construction of recombinant adenoviruses containing expression cassettes in the E1 and/or E3 regions. Using a versatile set of restriction enzymes, the cassettes are cloned into small bacterial vectors and subsequently introduced into large plasmids containing the adenoviral sequences. Two positive selection markers facilitate the recovery of a cosmid containing a copy of the sequence of the recombinant adenovirus. The resulting cosmid is transfected into 293 or 911 cells in order to rescue the virus. Importantly, the method does not require any recombination event, either in E. coli or in mammalian cells. The entire procedure can generate viral plaques in 12 days. Gene Therapy (2000) 7, 80-87.

Adenoviridae↗
PubMed2001

Expression of vhs and VP16 during HSV-1 helper virus-free amplicon packaging enhances titers.

Recently developed helper virus-free methods of herpes simplex virus (HSV) amplicon vector packaging provide stocks that are virtually devoid of the cytotoxic component normally associated with traditional helper virus-based packaging methods. These approaches involve cotransfection of amplicon plasmid DNA with either a five-cosmid set or a bacterial artificial chromosome (BAC) that contains the HSV genome without its cognate pac signals. Helper virus-free amplicon packaging produces low-titer stocks (<10(5) expressing particles/ml) that exhibit a high frequency of pseudotransduction. In an effort to enhance amplicon titers, we introduced in trans a genomic copy of the virion host shutoff (vhs) protein-encoding gene UL41 into both cosmid- and BAC-based packaging strategies. Cotransfection of this plasmid with the amplicon and packaging reagents results in a 10-fold higher amplicon titer, and stocks that do not exhibit the pseudotransduction phenomenon. To further enhance packaging efficiency, the HSV transcriptional activator VP16 was introduced into packaging cells 1 day before the packaging components. Pre-loading of packaging cells with VP16 led to an additional enhancement of amplicon titers, an effect that did not occur in the absence of vhs. Increased helper virus-free amplicon titers resulting from these modifications will make in vivo transduction experiments more feasible.

Animals↗
PubMed1997

Mosaicism of the 5q deletion as assessed by interphase FISH is a common phenomenon in MDS and restricted to myeloid cells.

Interstitial deletion of chromosome 5q is a common cytogenetic abnormality observed in MDS. We have used fluorescence in situ hybridization (FISH) to determine accurately the percentage of cytogenetically abnormal peripheral blood cells. YAC and cosmid probes localized to chromosome 5q were hybridized to interphase nuclei from purified polymorphonuclear cells (PMNs) from six MDS patients with chromosome 5 deletions. Per patient, 25-67% of the cells exhibited one signal for the 5q31-q33 specific probes IL-4, D5S207 and c-fms. This percentage was constant for the various probes utilized for each patient. Hybridization of the same probes to PMNs from healthy individuals and hybridization of probes (D5S39 and D5S498) localized outside the deleted segments to PMNs of the patients, resulted in 90-95% nuclei with two signals. In addition, FACS-purified peripheral blood cells were investigated by FISH using the IL-4 cosmid. This demonstrated that the hybridization pattern in monocytes was similar to that observed in PMNs, whereas T-lymphocytes showed no loss of signals. These results indicate that a subfraction of the myeloid progenitor cells have acquired the 5q deletion.

Adult↗