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At least 1,459 records · Page 81Linked to original sources
PubMed1999

A fine integrated map of the SPG4 locus excludes an expanded CAG repeat in chromosome 2p-linked autosomal dominant spastic paraplegia.

Autosomal dominant hereditary spastic paraplegia (AD-HSP) is a genetically heterogeneous disorder characterized by progressive spasticity of the lower limbs. A major locus (SPG4) causing AD-HSP in about 40% of the families was mapped to chromosome 2p. The analysis of six SPG4-linked AD-HSP families using the RED procedure previously showed the expansion of a CAG repeat in affected individuals. To identify the gene responsible for this form of HSP, we have constructed a 3.5-Mb YAC contig flanked by loci D2S400 and D2S367, have subcloned five of these YACs spanning the candidate region into cosmids, and screened these cosmid libraries for the presence of CAG repeat sequences. Four CAG repeats have been identified but none of them is expanded in 26 patients from 13 SPG4-linked AD-HSP families. A gene map comprising 21 transcripts was established using expressed sequence tags (ESTs) assigned previously to this region of 2p21-p22 with radiation hybrid panels GeneBridge 4 and G3. Full-length cDNAs corresponding to the 14 ESTs mapping to the SPG4 interval flanked by loci D2S352 and D2S2347 were isolated and sequenced. None contains a CAG repeat in its coding sequence. Finally, we have assembled a BAC contig composed of 37 clones that were also screened for the presence of CAG repeats; this failed to detect additional repeats to those identified on YACs.

Chromosomes, Bacterial↗
PubMed2000

Molecular cytogenetic evaluation in a patient with a translocation (3;21) associated with blepharophimosis, ptosis, epicanthus inversus syndrome (BPES).

Blepharophimosis, ptosis, epicanthus inversus syndrome type I (BPES; OMIM 110100) is an autosomal dominant disorder affecting craniofacial development and ovarian function. We have identified a patient with BPES who carried a de novo reciprocal translocation [46, XX,t(3;21)(q23;q22.1)]. Fluorescence in situ hybridization analysis at band 3q23 using probes derived from BAC 175G20 (Research Genetics), PACs 108L15 and 169C10 (RPCI1), and cosmids AC174D4, AC68D3, AC44F5, and AC125C5 (Lawrence Livermore National Laboratory) was performed. The patient's breakpoint was found to lie within the overlapping region of the BAC and PACs but centromeric to all the cosmids. However, a 10.5-kb BamHI-digested fragment, common to the BAC and PAC clones, was shown to cross the breakpoint. The results have placed our patient's breakpoint proximal to that of the previously reported patient [46,XY,t(3;4)(q23;p15.2)] and within a 10.5-kb interval. This is the second patient in which a breakpoint was refined by molecular cytogenetics. Our findings emphasize the significance of this region for BPES.

Adult↗
PubMed2002

Physical and transcript map of a 2-Mb region in Xp22.1 containing candidate genes for X-linked mental retardation and short stature.

Genetic loci for several diseases, including X-linked nonspecific mental retardation and short stature, have been mapped to Xp22.1. In spite of the recent publications of two draft sequences for the human genome, this region seems to be largely unmapped and unsequenced. Here we report an integrated physical and transcript map of approximately 2-Mb from DXS8004 to DXS365. Using sequence tagged site (STS)-content mapping and chromosome walking, we assembled a genomic clone contig of 54 BACs and one cosmid with an estimated 4.5-fold coverage of this region. The minimum tiling path consists of 23 BACs and one cosmid. Onto this contig, we mapped 30 new STSs derived from the unique end-sequences of the BACs, three expressed sequence tags, five genes, and seven CpG islands. This integrated map provides a unique resource for the positional cloning of candidate disease genes mapping to Xp22.1 and is therefore of value for the completion of the genomic sequence of this region.

Base Sequence↗
PubMed1996

IRS-Bubble PCR: An Effective Method for Representative Amplification of Human Genomic DNA Sequences from Complex Sources

A rate-limiting step in the analysis of large segments of genomic DNA is the generation of a set of representative short single-copy sequences that can be used for development of fine-structure maps. In direct response to this need, we have developed interspersed repetitive DNA sequence (IRS)-bubble PCR. IRS-bubble PCR was designed to amplify the human DNA content of somatic cell hybrids, yeast artificial chromosomes (YACs), BACs, PACs, cosmids, and lambda phage and to result in greater complexity and representation than standard inter-IRS PCR. Here, we describe the application of IRS-bubble PCR to the generation of complex clone libraries for targeted STS development and the construction of robust hybridization probes for FISH mapping, "chromosome painting," or the assembly of cosmid contigs representing the human DNA content of somatic cell hybrids or YACs.

Journal Article↗
PubMed1995

Self-transmissible nif plasmid (pEA9) of Enterobacter agglomerans 339: molecular cloning and evidence for the existence of similar nif clusters on dissimilar plasmids in Enterobacter strains.

A cosmid library was generated to the 200-kb self-transmissible nif plasmid pEA9 isolated from Enterobacter agglomerans 339. The cosmid clone identified to contain the complete nif cluster was used to determine the nif gene organization and the physical map. The restriction pattern and nif gene organization of this nif cluster showed remarkable similarities to the nif cluster identified on the 110-kb plasmid pEA3 of Enterobacter agglomerans 333. Nucleotide sequence of several randomly selected regions of the nif cluster of pEA9 showed 96% similarity when compared to the known sequences of the nif cluster of pEA3. However, the homology ended abruptly at the flanking regions of the nif clusters and no similarity could be detected with the rest of the DNA of these plasmids. This reveals the existence of similar nif clusters on dissimilar plasmids, implying the horizontal transfer of the entire nif gene cluster.

Bacterial Proteins↗
PubMed1998

Conjugal transfer of plasmid DNA from Escherichia coli to enterococci: a method to make insertion mutations.

Shuttle vector pAT18 was transferred by conjugation from Escherichia coli S17-1 to Enterococcus faecalis OG1RF and Enterococcus faecium SE34. Transfer was mediated by the transfer functions of plasmid RK2 in E. coli S17-1 and the origin of conjugal transfer (oriT) located on pAT18. The oriT sequence was then inserted into two plasmids to generate vectors pTEX5235 and pTEX5236. These two vectors cannot replicate in gram-positive bacteria and can be used to make insertion mutants in gram-positive bacteria. An internal sequence from an autolysin gene of E. faecalis OG1RF was cloned into pTEX5235 and transferred by conjugation from E. coli S17-1 to E. faecalis OG1RF. The plasmid was found to integrate into the chromosome of OG1RF by a single crossover event, resulting in a disrupted autolysin gene. A cosmid carrying the pyrimidine gene cluster from E. faecalis, with a transposon insertion in pyrC, was also transferred from E. coli S17-1 to E. faecalis OG1RF. After selection for the transposon, it was found to have recombined into the recipient chromosome by a double crossover between the cosmid and the chromosome of OG1RF. This resulted in a pyrC knockout mutant showing an auxotrophic phenotype.

Conjugation, Genetic↗
PubMed1995

Identification of essential trans-acting regions required for DNA replication of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus: lef-1 is an essential replication gene.

A transient replication assay for the identification of baculovirus genes that are essential for replication of an origin-containing reporter plasmid was established for the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV). Using a replication origin located on the OpMNPV HindIII-N fragment, we identified a subset of cosmids and plasmids from an OpMNPV cosmid library that was able to supply all the essential trans-acting factors and support replication of the origin-containing plasmid in uninfected Lymantria dispar cells. However, this limited set of DNA's was unable to support replication of a second origin-containing plasmid derived from a different region of the OpMNPV genome. Replication analysis of deletion clones of the HindIII-N fragment led to the identification of a gene, late expression factor 1 (lef-1), that is essential for the transactivation of DNA replication in this system. Transcriptional analysis of lef-1 mapped both early and late transcripts of about 1.75 kb. A motif characteristic of nucleoside triphosphate-binding sites present in the carboxy-terminal region of AcMNPV Lef-1 is not conserved in OpMNPV Lef-1.

Amino Acid Sequence↗
PubMed1984

Analysis of CAD gene amplification using a combined approach of molecular genetics and cytogenetics.

CAD is a multifunctional protein which catalyzes the first three steps of de novo uridine biosynthesis. Rodent cells resistant to PALA, a specific inhibitor of the ATCase activity of CAD, overproduce the CAD protein and CAD mRNA as a direct result of the amplification of the CAD gene. In order to study the mechanism of CAD gene amplification, a functional Syrian hamster CAD gene was inserted into a cosmid vector using molecular cloning techniques. The cloned genes were assayed for biological function by fusing CAD-deficient Chinese hamster ovary (CHO) cell mutants with protoplasts of E. coli containing the CAD cosmids. Two clones with functional CAD genes were isolated and shown to contain inserts 40 and 45 kb long. The cloned genes could also be introduced into wild type CHO cells by selecting for cells which became resistant to high PALA concentrations in a single step. Transformations of mutant and wild type CHO cells contained multiple active copies of the donated Syrian hamster CAD genes in addition to their endogenous CHO CAD genes. The cloned genes in all transformants analyzed are integrated into host cell chromosomes at single locations defined by in situ hybridization. Independently isolated transformants contain the donated genes in different chromosomes. Co-transformation of CHO cells with two different genes by protoplast fusion is also shown to be possible.

Animals↗
PubMed1993

Isolation of a delta 6-desaturase gene from the cyanobacterium Synechocystis sp. strain PCC 6803 by gain-of-function expression in Anabaena sp. strain PCC 7120.

The enzyme delta 6-desaturase is responsible for the conversion of linoleic acid (18:2) to gamma-linolenic acid (18:3 gamma). A cyanobacterial gene encoding delta 6-desaturase was cloned by expression of a Synechocystis genomic cosmid library in Anabaena, a cyanobacterium lacking delta 6-desaturase. Expression of the Synechocystis delta 6-desaturase gene in Anabaena resulted in the accumulation of gamma-linolenic acid (GLA) and octadecatetraenoic acid (18:4). The predicted 359 amino acid sequence of the Synechocystis delta 6-desaturase shares limited, but significant, sequence similarity with two other reported desaturases. Analysis of three overlapping cosmids revealed a delta 12-desaturase gene linked to the delta 6-desaturase gene. Expression of Synechocystis delta 6- and delta 12-desaturases in Synechococcus, a cyanobacterium deficient in both desaturases, resulted in the production of linoleic acid and gamma-linolenic acid.

Amino Acid Sequence↗
PubMed1996

Desferrioxamine-dependent iron transport in Erwinia amylovora CFBP1430: cloning of the gene encoding the ferrioxamine receptor FoxR.

Iron deprivation of Erwinia amylovora CFBP1430, a species causing fire blight on Pomoïdeae, was shown to induce the production of siderophores of the desferrioxamine (dfo) family and two outer membrane polypeptides with apparent molecular weight of about 70 and 80 kDa, respectively. Cyclic dfo E was characterized as the major metabolite. Phage MudIIpR13 insertional mutagenesis and screening on CAS-agar medium yielded three dfo non-producing and one overproducing clones. These clones failed to grow in the presence of the Fe(III) chelator EDDHA and were determined further as dfo and ferrioxamine transport negative mutants, respectively. The transport mutant which appeared to lack the 70 kDa polypeptide in the outer membrane allowed the purification of dfo E. Growth under iron limitation of dfo negative mutants was stimulated with ferrioxamine E and B but not with other ferrisiderophores tested. The host DNA sequence flanking the left terminal part of the MudIIpR13 prophage responsible for the transport mutation was cloned and used to probe a parental gene library by DNA-DNA hybridization. Two recombinant cosmids restoring the transport mutation to normal were identified. Both cosmids also conferred the ability to utilize ferrioxamine B and E as iron sources on a FhuE- mutant of Escherichia coli. This correlated with the production of an additional polypeptide of 70 kDa in the outer membrane of E. coli transconjugants, thus confirming that this protein serves the ferrioxamine receptor function (FoxR) in E. amylovora.

Bacterial Outer Membrane Proteins↗
PubMed1993

Organization and structure of the H-2M4-M8 class I genes in the mouse major histocompatibility complex.

We have cloned and characterized five new class I genes from the M region at the distal end of the H-2 complex of the BALB/c mouse. M4, M5, and M6 are clustered on two overlapping cosmids, and M7 and M8 are located on another cosmid together with the previously cloned M1 gene, to which they are most closely related. M4, M6, and M7 are full-length class I genes (exons 1 through 5 were identified) but with stop codons or frameshifts that mark them as pseudogenes, and only exons 4 and 5 remain of M8. M5 has complete open reading frames in exons 1 through 5 and intact splice signals; it has the potential to encode a divergent class I major histocompatibility molecule, but no transcripts were found. These genes provide probes for studying the evolution of class I genes in rodents and for the mapping and cloning of genes at the end of the distal inversion in t haplotype chromosomes.

Amino Acid Sequence↗
PubMed1990

The human homeobox gene HOX7 maps to chromosome 4p16.1 and may be implicated in Wolf-Hirschhorn syndrome.

A cosmid containing the human sequence (HOX7) homologous to the mouse homeogene Hox-7 was isolated from a genomic cosmid library. There is only one highly conserved homologous gene in the human genome. The C-terminal two-thirds of the HOX7 homeobox DNA sequence has been determined; there are no predicted amino acid changes from the mouse sequence. Data from mouse/human hybrid cell lines show that HOX7 maps to human chromosome 4p16.1, a region that is syntenic with part of mouse chromosome 5, the site of the murine Hox-7 gene. Analysis of chromosomes from two patients with Wolf-Hirschhorn syndrome, which is characterised by profound dysmorphologies, indicates that the HOX7 locus is deleted. Although not all Wolf-Hirschhorn syndrome patients analysed were deleted for HOX7, the combination of positional data and functional correlation with mouse expression implicates HOX7 as a candidate gene for this syndrome.

Animals↗
PubMed1991

MHC gene Q8/9d of the BALB/cJ mouse strain cannot encode a Qa-2,3 class I antigen.

We have determined the nucleotide sequence of Q8/9d gene of the BALB/c strain of mice, isolated from Steinmetz's cosmid library. As for all other class I genes of the Qa region, the Q8/9d gene spans approximately 4.7 kilobases (kb) and consists of seven exons and six introns. A seven bases deletion in exon 3 results in the occurrence of an early termination codon. Thus the Q8/9d gene cannot encode a normal class I protein. Comparison of the nucleotide sequence of the Q8/9d gene with that of other class I MHC genes revealed a stronger homology to Q7 and Q8 than to K, D, L, TL, and other Q genes. However, the gene cannot originate from a mere fusion between Q8 and Q9 genes except if the ancestor to putative Q8d was markedly different from the present Q8b gene. Using polymerase chain reaction (PCR) technology, we have confirmed the presence of a Q8/9 gene, identical to that present in cosmid 46.1, in the genome of BALB/cJ (Qa-2low). Finally, it has been reported that cDNA clone 94-A, which codes for a Qa-2 antigen, could derive from a transcript of gene Q8/9d. The nucleotide sequences of gene Q8/9d and of cDNA clone 94-A are distinctly different in their 5' regions, in spite of an almost perfect matching in their 3' regions. Thus, clone 94-A cannot derive from an mRNA transcribed from the Q8/9d gene.

Animals↗
PubMed1991

Relationship between tryptophan biosynthesis and indole-3-acetic acid production in Azospirillum: identification and sequencing of a trpGDC cluster.

Screening the tryptophan (Trp)-dependent indole-3-acetic acid (IAA) production of different Azospirillum species revealed that A. irakense KA3 released 10 times less IAA into the medium than A. brasilense Sp7. A cosmid library of strain Sp7 was transferred into A. irakense KA3 with the aim of characterizing genes involved in IAA biosynthesis. Trp-dependent IAA production was increased in two transconjugants which both contained an identical 18.5 kb HindIII fragment from Sp7. After Tn5 mutagenesis, cosmids carrying Tn5 insertions at 36 different positions of the 18.5 kb fragment were isolated and transferred into strain KA3. IAA production by the recipient strains was screened by HPLC. The Tn5 insertions of 4 clones with decreased IAA production were mapped on a 2 kb SalI-SphI fragment. Recombination of Tn5 insertions at this locus into the genome of strain Sp7 led to Trp auxotrophic mutants. A 5.2 kb EcoRI-SalI fragment including the 2 kb SalI-SphI fragment was sequenced and six open reading frames were identified. Three of them were clustered and their deduced amino acid sequences showed significant similarity to TrpG, TrpD and TrpC, which are enzymes involved in tryptophan biosynthesis. One of the remaining open reading frames probably encodes an acetyltransferase. The region responsible for the enhanced Trp-dependent IAA production in strain KA3 corresponded to trpD, coding for the phosphoribosyl anthranilate transferase.

Acetyltransferases↗
PubMed1981

Location of nodulation and nitrogen fixation genes on a high molecular weight plasmid of R. meliloti.

R. meliloti strain 41 (Rm41) was shown to harbour two indigenous plasmids with molecular weights of 140 Mdal (pRme41a) and more than 300 Mdal (pRme41b), respectively. Using a heat-treatment procedure, derivatives of Rm41 defective in nodulation (Nod-) or nitrogen fixation (Fix-) have been readily obtained. In some Nod- mutants the deletion of a segment of plasmid pRme41b was found. Based on the demonstrated homology between the nitrogen fixation (nif) genes of Klebsiella pneumoniae and of R. meliloti the Rhizobium nif region has been cloned into the cosmid vector pHC79, then recloned into pBR322 and the restriction map of the nif region has been determined. 32P-labelled nick-translated probe prepared from the cloned nif DNA fragment hybridized to pRme41b of Rm41 but for most Nod- mutants this hybridization was not detected. Hybridization of a cosmid containing Rm41 DNA to total DNA digest from the wild-type bacterium and from a series of Nod- mutants revealed that at least a 2 kb DNA fragment including the nif structural genes was missing from most of the Nod- mutants. These results, together with the genetic analyses of these symbiotic mutations suggest that some nod and fix genes are located on pRme41b.

Chromosome Mapping↗
PubMed1989

Two type II keratin genes are localized on human chromosome 12.

Human genomic DNA containing two type II keratin genes, one coding for keratin 1 (K1, a 68-kD basic protein) and another closely linked type II gene 10-15 kb upstream (K?, gene product unknown), was isolated on a single cosmid clone. EcoRI restriction fragments of the cosmid were subcloned into pGEM-3Z, and specific probes comprising the C-terminal coding and 3' noncoding regions of the two genes were constructed. The type II keratin genes were localized by in situ hybridization of the subcloned probes to normal human lymphocyte chromosomes. In a total of 70 chromosome spreads hybridized with the K? probe (gHK?-3', PstI, 800 bp), 36 of the 105 grains observed were on chromosome 12, and 32 of these were clustered on the long arm near the centromere (12q11-13). In 100 labeled metaphases hybridized with the K1 probe (gHK1-3', BamHI-PstI, 2100 bp), 53 grains localized to chromosome 12 and 46 of these were found in the same region (q11-13). Therefore, both the gene for human keratin 1, a specific marker for terminal differentiation in mammalian epidermis, and another closely linked unknown type II keratin gene (K?, 10-15 kb upstream of K1) are on the long arm (q11-13) of human chromosome 12.

Cells, Cultured↗
PubMed1992

The PYR1 gene of the plant pathogenic fungus Colletotrichum graminicola: selection by intraspecific complementation and sequence analysis.

A spontaneous uridine-requiring auxotroph of Colletotrichum graminicola was recovered by selection for resistance to 5-fluoro-orotic acid. The auxotroph lacked orotate phosphoribosyl transferase (OPRTase) and was complemented with a clone from a cosmid library of C. graminicola DNA. A 3.1 kb HindIII-SalI fragment was subcloned from the cosmid and it could efficiently transform the auxotrophic strain to uridine prototrophy and integrate by site-specific recombination. This DNA fragment contains an open reading frame that is similar to OPRTase genes of the fungi Sordaria macrospora, Trichoderma reesei, Podospora anserina, and Saccharomyces cerevisiae. Based on the sequence similarities and the ability to restore uridine prototrophy, we conclude that the fragment contains the C. graminicola gene for OPRTase, which we have named PYR1. Our results demonstrate that cloning by complementation is feasible in C. graminicola, that the gene for OPRTase from C. graminicola can be useful as a selectable marker in transformation of the fungus, and that the OPRTase gene product is similar to OPRTase from other fungi.

Amino Acid Sequence↗
PubMed1995

Physical mapping of sequences homologous to an endogenous retrovirus LTR on human chromosome 19.

The human genome contains multiple copies of sequences related to the HERV-K family of endogenous retroviruses, homologous to the B-type mouse mammary tumour virus. A DNA fragment closely resembling an HERV-K long tandem repeat (LTR) was detected in a library of hncDNA clones enriched for sequences from human chromosome 19. Sites showing homology to the sequence of this fragment have been identified on human chromosome 19 by hybridization to previously mapped chromosome 19 cosmids. Thus the distribution of LTR sequences on a specific human chromosome has been mapped for the first time. We estimate the total number of such sites on human chromosome 19 to be at least 110. Many of these sites are located in the vicinity of known genes. The precise localizations (to specific cosmids) of LTR-homologous sequences on chromosome 19 can serve as a reference source and will automatically provide further insight into LTR-gene relationships as new genes are mapped onto the chromosome.

Animals↗