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PubMed1998

High resolution physical mapping and identification of transcribed sequences in the Down syndrome region-2.

The identification and mapping of genes within the Down syndrome region is an important step toward a complete understanding of the pathogenesis of this disorder. The objective of the present work is to identify and map genes within the Down syndrome region-2. Chromosome 21 cosmid clones corresponding to "cosmid pockets" 121-124 have been first used as a starting material for generation of a single high resolution integrated cosmid/PAC contig with full EcoRI/SmaI restriction map. The integrated contig has been further anchored to genetic and physical maps through the positioning of 6 markers in the following order: ACTL5-D21S3-684G2T7-D21S71-D21S343-D21S 268. The entire contig covers 342 kb of the Down syndrome region-2 of chromosome 21. Subsequently, we have isolated, identified, and mapped four novel cDNAs which we have named N143, N144, CHD/333, and 90/3H1 and a potentially transcribed genomic sequence (E05133T7). Additionally, we have accurately located a previously described gene, the WRB gene, between the markers ACTL5-D21S268 within this Down Syndrome Region-2.

Chromosome Mapping↗
PubMed1993

An intraspecific gene duplication polymorphism of the urate oxidase gene of Drosophila virilis: a genetic and molecular analysis.

Nineteen strains of Drosophila virilis from diverse geographic locations were examined by genetic and molecular analyses, revealing (a) 12 strains with a single copy of the urate oxidase (UO) gene per haploid genome and (b) 7 strains with a tandem duplication of the UO locus. The D. virilis strains with the UO duplication appear to have identical restriction maps of this region, implying either a single origin for the duplication or several similar events occurring at a hot spot. On the basis of the location of the duplication breakpoints and the restriction sites flanking these breakpoints, this duplication probably arose through nonhomologous recombination involving either a breakage and rejoining event or replication slippage. because documented cases of intraspecific gene duplication polymorphism are rare, the D. virilis UO duplication will be useful in identifying the molecular event giving rise to a gene duplication.

Animals↗
PubMed1988

Plasmid DNA from the acetotrophic methanogen Methanosarcina acetivorans.

Nine acetotrophic and three methylotrophic strains of methane-producing bacteria were screened for the presence of plasmid DNA. Plasmids were detected in three marine isolates, including Methanosarcina acetivorans. All three plasmids appeared to be similar based on size and restriction site analyses. The plasmid from M. acetivorans, designated pC2A, was approximately 5.1 kilobase pairs in size and was estimated to be present in a low copy number of six plasmids per genome. Multimers were also observed. A restriction map was constructed. The function of this plasmid is cryptic.

DNA, Bacterial↗
PubMed1992

DNA rearrangements in the alpha 5(IV) collagen gene (COL4A5) of individuals with Alport syndrome: further refinement using pulsed-field gel electrophoresis.

Alport syndrome (AS), an X-linked kidney disorder, has been shown to be caused by mutations in the gene for the alpha 5-chain of type IV collagen (COL4A5), which maps to Xq22. On the basis of the results of conventional Southern blot analysis of AS patient DNAs, we employed pulsed-field gel electrophoresis to characterize further three gene rearrangements at the 3'-end of alpha 5(IV). We were able to construct long-range restriction maps for all three of these patients and deduce the extent and nature of each rearrangement. One of these mutations is a 450-kb simple deletion that includes 12 kb of the alpha 5(IV) gene. A second mutation has been shown to be a direct duplication of 35 kb of alpha 5(IV) genomic DNA, and a third mutation involves a complex insertion/deletion event resulting in an overall loss of 25 kb.

Chromosome Mapping↗
PubMed1992

[Isolation, characterization and mapping of the N15 phasmid insertion mutations].

Prophage of N15 temperate bacteriophage is stably maintained in Escherichia coli lysogens as a 46.33 kb linear plasmid. Using different transposons we obtained 18 insertion mutants of the N15 plasmid prophage. They were analysed for plaque formation ability, stability of the plasmid state and lysogenic conversion. Restriction mapping of the insertions allowed us to localize on the map the regions necessary for lytic growth and to map the lysogenic conversion gene. A recombinant phage encoding two antibiotic resistance genes was obtained. The phage contains an additional 4.77 kb DNA fragment (over 10% of the N15 genome).

Bacteriophages↗
PubMed1989

Cystic fibrosis: diagnostic testing and the search for the gene.

Cystic fibrosis (CF) is a lethal genetic disorder inherited as an autosomal recessive at a frequency of about 1/2000 in Caucasian populations. A DNA marker genetically linked to CF was identified through a collaborative effort by random screening with a collection of RFLP markers on a set of CF families. The marker (CRI-L917) was mapped to chromosome 7. Construction of a genetic linkage map spanning the entire chromosome has led to the identification of a subset of 11 markers close to and flanking the CF locus. Using techniques of pulsed-field gel electrophoresis, which allow very large DNA fragments to be separated, we used seven probes to generate a long-range restriction map covering 12 million base pairs surrounding the CF locus. Information from the map is being used to isolate new probes closer to the CF gene. Methods being developed will allow candidate genes to be tested for their ability to correct defects in ion transport in cultured CF cells.

Chromosome Mapping↗
PubMed1992

Adhesive properties conferred by the plasminogen activator of Yersinia pestis.

A genomic library of Yersinia pestis EV76c created in a cosmid vector was screened for clones capable of binding type IV collagen. An unexpectedly high number of such clones was observed. One recombinant plasmid was selected for further study, and the locus controlling collagen binding was mapped by subcloning, transposon mutagenesis and exonuclease digestion. The outer-membrane protein profiles of transposon insertion mutants were correlated with phenotype to implicate a 36 kDa polypeptide in type IV collagen binding. Fine substructure restriction mapping and limited DNA sequence analysis showed the cloned locus to be identical to the locus (pla) for the plasminogen activator, previously characterized genetically and biochemically. The pla locus is resident on a 9.5 kb plasmid in wild-type Y. pestis strains. Curing of this plasmid resulted in negligible reduction in collagen-binding capacity, implying the existence of a chromosomally located determinant for collagen binding. The affinity of the plasminogen activator for collagen was relatively weak. When the cloned pla locus was introduced into E. coli, it conferred upon the cell the ability to bind to cells from a number of cell lines. Binding to glycolipids separated by thin-layer chromatography demonstrated that the receptor was a member of the globo-series of glycolipids. Since it has been reported that mutation of pla dramatically reduces virulence, we propose that this hitherto undescribed function of the gene product could contribute to the biological activities necessary for full virulence.

Bacterial Adhesion↗
PubMed1993

Rapid detection and prevalence of cholesteryl ester transfer protein deficiency caused by an intron 14 splicing defect in hyperalphalipoproteinemia.

A deficiency of plasma cholesteryl ester transfer protein (CETP) is one of the genetic causes of increased serum high density lipoprotein (HDL)-cholesterol levels (hyperalphalipoproteinemia). A splicing defect (G-->A mutation) at the +1 position of intron 14 of the human CETP gene is a common mutation in the Japanese CETP deficiency. A rapid screening method for the splicing defect by means of primer-specified restriction map modification was described. The frequency of the mutation in hyperalphalipoproteinemia was determined, and its frequency in the general population was estimated. During polymerase chain reaction (PCR) with a modified primer, a novel NdeI restriction endonuclease site was created from the mutated allele in the PCR products, which could be visualized after electrophoresis of the digested products. As a result, 21 of 121 unrelated hyperalphalipoproteinemic subjects with HDL-cholesterol > or = 60 mg/dl (1.55 mmol/l), were found to have the G-->A mutation. Of the 21 individuals, 8 were found to be homozygous for the mutation. Allele frequency of the mutation was 1.5% (1/68), 2.8% (2/72), 7.1% (4/56), and 47.8% (22/46) in the groups with HDL-cholesterol levels of 60-79 mg/dl, 80-99 mg/dl, 100-119 mg/dl, and > or = 120 mg/dl, respectively. Based on the percentage of the area under the computed normal distribution curve of serum HDL-cholesterol, the frequency of the mutated allele in the general population was estimated to be 0.81% from the present results. This rapid detection method facilitates large-scale screening of CETP deficiency caused by the splicing defect. The mutation was frequent in Japanese subjects with hyperalphalipoproteinemia, especially in the group with HDL-cholesterol > or = 120 mg/dl.

Adult↗
PubMed1990

Selective cloning of B cell hybridoma-specific rearranged immunoglobulin gene loci using the polymerase chain reaction.

The use of conventional DNA cloning procedures to obtain productively rearranged Ig genes from B cell hybridomas for structure/function analysis of immunoglobulins is tedious and time-consuming. Here we describe a procedure based on PCR which permits rapid, selective isolation of DNA segments containing individual hybridoma-specific Ig gene rearrangements. The method, an adaptation of the so-called 'inverted PCR' technique (IPCR), can be applied most efficiently to specific genes where a preliminary restriction map is available from Southern blot analysis of the hybridoma genomic DNA. To achieve amplification of a given rearranged Ig locus, small amounts of total hybridoma DNA are digested to completion with a chosen restriction endonuclease and the fragments circularised by DNA ligase. Cleavage of the DNA circles using a second restriction enzyme, chosen specifically to cut 3' to a rearranged V-(D)-J exon, leads to linear DNA segments where the rearranged gene is now flanked by segments of known nucleotide sequence derived originally from the 3' region of the Ig H or L chain gene locus. This permits the selection of oligonucleotides that provide convergent primers for specific amplification of DNA segments containing the required gene rearrangement. Amplified DNA fragments can be cloned and rapidly characterised by sequence analysis.

Animals↗
PubMed1994

The ARG4 gene of Candida albicans.

The DNA sequence of a Candida albicans genomic fragment known to complement the arginine mutation designated arg57 in strain 1006 contains an ORF of 1404 nucleotides (nt) predicting a protein of 468 amino acids (aa). Database searches indicated that the deduced protein shares 75% identity and 85% similarity with the ARG4 protein of Saccharomyces cerevisiae. Analysis of the percent aa identity between C. albicans and S. cerevisiae sequences included in available databases suggested these values are within the range expected for biosynthetic enzymes from the two organisms which share similar function. Experiments to isolate C. albicans ARG4 by complementation in an arg4 strain of S. cerevisiae yielded a plasmid (pARG4-1) with a restriction map identical to that of the sequenced clone. From these data, we conclude that the gene previously designated ARG57 is in fact ARG4 encoding the enzyme argininosuccinate lyase (ASL). These results were unexpected, since ARG57 had been localized to chromosome 7, while a mutation causing an ASL deficiency had been linked to ade1, which is on chromosome R. Transformation of C. albicans strains with pARG4-1 indicated it complemented the arginine auxotrophy in strains TMSU221 and 1435, a derivative of 1006. Examination of commonly utilized C. albicans arginine auxotrophs by spheroplast fusion analysis indicated these strains comprise two complementation groups: one consisting of 1006 and TMSU221, which are arg4, and the other of A642, hOG318, hOG357, FC18-6 and WC-5-4, which possess an undefined defect in the arginine biosynthetic pathway which we designate arg100.

Amino Acid Sequence↗
PubMed1989

Chromosomal localization of DBL oncogene sequences.

The DBL oncogene was generated by rearrangements involving three discontinuous regions of the human genome. Analyses of panels of human X rodent somatic cell hybrids demonstrated that the DBL proto-oncogene located on the X chromosome (just proximal or distal to bands q26-27.2) underwent recombination at its 5' and 3' ends with sequences derived from chromosomes 3 (p13q-ter) and 16 (p13-q22), respectively. DBL was localized to chromosome Xq27-q28 by in situ hybridization. Another oncogene, MCF2, was previously shown to contain sequences derived from Xq27 as well. Comparison of the restriction maps and nucleotide sequences of genomic and cDNA clones representing the chromosome X-specific sequences of the DBL oncogene and MCF2, taken together with their chromosomal localization, indicates that these oncogenes were derived from the same genetic locus.

Animals↗
PubMed1998

Genomic characterization of the coding region of the human type II 5'-deiodinase gene.

Type II 5'-Deiodinase (5'DII) is a key element in the maintenance of peripheral thyroid hormone homeostasis through the regulation of local T4 to T3 conversion in pituitary, brain, brown adipose tissue and placenta. The cDNA containing the coding region of the human 5'DII (HDII) has been recently cloned from infant brain. In the present paper we report the genomic structure, chromosomal localization and restriction map of the coding region of HDII. The presence of a single intron located at codon 75 was demonstrated using a PCR-based strategy; the exon-intron junctions were then cloned and partially sequenced. Chromosomal localization was performed by radiation hybrid mapping. This study demonstrated that the entire coding region of the HDII gene is contained in two exons spliced at codon 75 by a 7.4 Kb intron and that the HDII chromosomal location is 14q24.3. These data will allow further studies of the role of HDII in the pathophysiology of thyroid homeostasis.

Base Sequence↗
PubMed1998

A knob-associated tandem repeat in maize capable of forming fold-back DNA segments: are chromosome knobs megatransposons?

A class of tandemly repeated DNA sequences (TR-1) of 350-bp unit length was isolated from the knob DNA of chromosome 9 of Zea mays L. Comparative fluorescence in situ hybridization revealed that TR-1 elements are also present in cytologically detectable knobs on other maize chromosomes in different proportions relative to the previously described 180-bp repeats. At least one knob on chromosome 4 is composed predominantly of the TR-1 repeat. In addition, several small clusters of the TR-1 and 180-bp repeats have been found in different chromosomes, some not located in obvious knob heterochromatin. Variation in restriction fragment fingerprints and copy number of the TR-1 elements was found among maize lines and among maize chromosomes. TR-1 tandem arrays up to 70 kilobases in length can be interspersed with stretches of 180-bp tandem repeat arrays. DNA sequence analysis and restriction mapping of one particular stretch of tandemly arranged TR-1 units indicate that these elements may be organized in the form of fold-back DNA segments. The TR-1 repeat shares two short segments of homology with the 180-bp repeat. The longest of these segments (31 bp; 64% identity) corresponds to the conserved region among 180-bp repeats. The polymorphism and complex structure of knob DNA suggest that, similar to the fold-back DNA-containing giant transposons in Drosophila, maize knob DNA may have some properties of transposable elements.

Base Sequence↗
PubMed1990

[Cloning of the Streptococcus thermophilus beta-galactosidase gene and its expression in Escherichia coli and Bacillus subtilis cells].

The beta-galactosidase gene from the chromosome of Streptococcus thermophilus, strain 6 kb, has been cloned on a vector plasmid pBR322. The corresponding gene has been found to be located on the Pst1 DNA fragment. The restriction map of this 6 kb fragment has been constructed. The shortening of the DNA fragment carrying the beta-galactosidase gene has been achieved by digestion of the recombinant derivative of pBR322 by the restriction endonuclease Sau3A under the conditions of incomplete hydrolysis. The obtained fragments have been cloned into the BamHI site in the berepliconed shuttle vector pCB20 for grampositive and gramnegative bacteria. The obtained recombinant plasmids contained the beta-galactosidase gene in the inserted fragments of different length. Expression of the cloned beta-galactosidase gene in Escherichia coli and Bacillus subtilis cells has been studied.

Bacillus subtilis↗
PubMed1991

D11S146 and BCL1 are physically linked but can be discriminated by their amplification status in human breast cancer.

Band q13 of chromosome 11 is frequently altered in a number of human cancers. We have undertaken physical mapping in this region, starting with D11S146, an anonymous 11q13 DNA fragment. This probe has been used by others as a landmark to locate MEN1, a locus of predisposition to multiple endocrine neoplasia. Long-range restriction mapping locates D11S146 within approximately 400 kb of the BCL1 translocation breakpoint involved in certain B-cell malignancies. BCL1 and two proto-oncogenes, INT2 and HST, were previously found to be coamplified in approximately 1/5 breast carcinomas. Although close to BCL1, D11S146 is present in less than 3/4 of these amplification units and delimits their centromeric boundary. Therefore, we propose that D11S146 defines two genetic regions. The centromeric region--PYGM/D11S146--contains MEN1. The telomeric one includes the D11S146/BCL1/INT2/HST area and is relevant to DNA amplification in carcinomas and to B-cell translocations.

Breast Neoplasms↗
PubMed1998

[Cloning the BR-C gene in Drosophila virilis].

In 1980, Belyaeva et al. found in Drosophila melanogaster the ecs (ecdysterone sensitivity) locus, later designated BR-C (Broad-Complex). This locus plays a key role in the reaction cascade of ecdysterone-regulated development. The gene BR-C was cloned by Chao and Guild (1986) and Belyaeva et al. (1987). To conduct "walking along the chromosome" in Drosophila virilis, cloned sequences of the BR-C gene from D. melanogaster were used as probes, hybridizing the start sites. A fragment of about 100 kb containing BR-C of D. virilis was cloned. A restriction map of the cloned region was created.

Animals↗
PubMed1999

Genomics via optical mapping. III: Contiging genomic DNA.

In this paper, we describe our algorithmic approach to constructing an alignment of (contiging) a set of restriction maps created from the images of individual genomic (uncloned) DNA molecules digested by restriction enzymes. Generally, these DNA segments are sized in the range of 1-4 Mb. The goal is to devise contiging algorithms capable of producing high-quality composite maps rapidly and in a scaleable manner. The resulting software is a key component of our physical mapping automation tools and has been used to create complete maps of various microorganisms (E. coli, P. falciparum and D. radiodurans). Experimental results match known sequence data.

Algorithms↗
PubMed1991

Isolation, characterization, and chromosomal mapping of mouse P450 17 alpha-hydroxylase/C17-20 lyase.

Cytochrome P450 17 alpha-hydroxylase/C17-20 lyase (P45017 alpha) catalyzes the conversion of C-21 steroids to C-19 steroids in gonads. A full-length mouse cDNA encoding P450 17 alpha was isolated from a mouse Leydig cell library and characterized by restriction mapping and sequencing. The predicted amino acid sequence has 83% homology to rat, 66% homology to human, and 62% homology to bovine P45017 alpha amino acid sequences. The protein is 507 amino acids in length, which is 1 amino acid shorter than the human protein and 2 amino acids shorter than the bovine protein. The structural gene encoding P450 17 alpha (Cyp17) was localized utilizing an interspecific testcross to mouse chromosome 19, distal to Got-1. Another cytochrome P450, P4502c (Cyp2c), also is located at the distal end of chromosome 19. CYP17, CYP2c, and GOT1 have been mapped to human chromosome 10, with CYP2C and GOT1 mapped to the distal region, q24.3 and q25.3, respectively. The data in the present study indicate conserved syntenic loci on mouse chromosome 19 and human chromosome 10 and predict that the structural gene encoding P45017 alpha will be found distal to GOT1 on human chromosome 10.

Amino Acid Sequence↗