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PubMed1997

YAC and cosmid contigs encompassing the Fukuyama-type congenital muscular dystrophy (FCMD) candidate region on 9q31.

Fukuyama-type congenital muscular dystrophy (FCMD), the second most common form of childhood muscular dystrophy in Japan, is an autosomal recessive severe muscular dystrophy associated with an anomaly of the brain. We had mapped the FCMD gene to an approximately 5-cM interval between D9S127 and D9S2111 on 9q31-q33 and had also found evidence for linkage disequilibrium between FCMD and D9S306 in this candidate region. Through further analysis, we have defined another marker, D9S172, which showed stronger linkage disequilibrium than D9S306. A yeast artificial chromosome (YAC) contig spanning 3,5 Mb, which includes this D9S306-D9S172 interval on 9q31, has been constructed by a combination of sequence-tagged site, Alu-PCR, and restriction mapping. Also, cosmid clones subcloned from the YAC were assembled into three contigs, one of which contains D9S2107, which showed the strongest linkage disequilibrium with FCMD. These contigs also allowed us to order the markers as follows: cen-D9S127-(approximately 800 kb)-D9S306 (identical to D9S53)-(approximately 700 kb)-A107XF9-(approximately 500 kb)-D9S172-(approximately 30 kb)-D9S299 (identical to D9S774)-(approximately 120 kb)-WI2269-tel. Thus, we have constructed the first high-resolution physical map of the FCMD candidate region. The YAC and cosmid contigs established here will be a crucial resource for identification of the FCMD gene and other genes in this region.

Chromosomes, Artificial, Yeast↗
PubMed1991

Isolation of a human DNA sequence which spans the fragile X.

To identify the sequences involved in the expression of the fragile X and to characterize the molecular basis of the genetic lesion, we have constructed yeast artificial chromosomes (YACs) containing human DNA and have screened them with cloned DNA probes which map close to the fragile site at Xq27.3. We have isolated and partly characterized a YAC containing approximately 270 kb of human DNA from an X chromosome which expresses the fragile X. This sequence in a yeast artificial ring chromosome, XTY26, hybridizes to the two closest DNA markers, VK16 and Do33, which flank the fragile site. The human DNA sequence in XTY26 also spans the fragile site on chromosome in situ hybridization. When a restriction map of XTY26, derived by using infrequently cutting restriction enzymes, is compared with similar YAC maps derived from non-fragile-X patients, no large-scale differences are observed. This YAC, XTY26, may enable (a) the fragile site to be fully characterized at the molecular level and (b) the pathogenetic basis of the fragile-X syndrome to be determined.

Chromosome Mapping↗
PubMed1994

Genetic analysis of type 5 capsular polysaccharide expression by Staphylococcus aureus.

Capsules are produced by over 90% of Staphylococcus aureus strains, and approximately 25% of clinical isolates express type 5 capsular polysaccharide (CP5). We mutagenized the type 5 strain Reynolds with Tn918 to target genes involved in CP5 expression. From a capsule-deficient mutant, we cloned into a cosmid vector an approximately 26-kb EcoRI fragment containing the transposon insertion. In the absence of tetracycline selection, Tn918 was spontaneously excised, thereby resulting in a plasmid containing 9.4 kb of S. aureus DNA flanking the Tn918 insertion site. The 9.4-kb DNA fragment was used to screen a cosmid library prepared from the wild-type strain. Positive colonies were identified by colony hybridization, and a restriction map of one clone (pJCL19 with an approximately 34-kb insert) carrying the putative capsule gene region was constructed. Fragments of pJCL19 were used to probe genomic DNA digests from S. aureus strains of different capsular serotypes. Fragments on the ends of the cloned DNA hybridized to fragments of similar sizes in most of the strains examined. Blots hybridized to two fragments flanking the central region of the cloned DNA showed restriction fragment length polymorphism. A centrally located DNA fragment hybridized only to DNA from capsular types 2, 4, and 5. DNA from pJCL19 was subcloned to a shuttle vector for complementation studies. A 6.2-kb EcoRI-ClaI fragment complemented CP5 expression in a capsule-negative mutant derived by mutagenesis with ethyl methanesulfonate. These experiments provide the necessary groundwork for identifying genes involved in CP5 expression by S. aureus.

Bacterial Capsules↗
PubMed1997

Human estrogen receptor-like 1 (ESRL1) gene: genomic organization, chromosomal localization, and promoter characterization.

Estrogen receptor-like 1a (ESRL1a; same as estrogen receptor-related orphan receptors, ERR1) belongs to a subfamily of the nuclear receptor superfamily. We have previously shown that human ESRL1a modulates estrogen responsiveness of the lactoferrin gene promoter in transiently transfected endometrial carcinoma RL95-2 cells. In this study, we cloned and characterized the human ESRL1 gene. Through the fluorescence in situ hybridization method, the ESRL1 gene was localized to the centromere region of chromosome 11q12. Partial sequencing, restriction mapping, and PCR analysis revealed that the ESRL1 gene consists of seven exons and is approximately 20 kb in length. We found that the smallest exon (exon 3) contains 117 bp and the largest exon (exon 7) has 1032 bp. The smallest intron (intron 5) is only 88 bp long and the largest intron (intron 2) is 8 kb long. All introns have the conserved GT and AG dinucleotides present at the donor and acceptor sites, respectively. Like the estrogen receptor, the highly conserved DNA-binding domain of hESRL1a is encoded by exon 2 and exon 3, and the intron/exon junctions (2 and 3) are well conserved between the two genes. Primer extension analysis revealed multiple transcription initiation start sites in human uterine (HeLa, HEC, and RL95-2) cell lines. However, one major initiation start site was found by RNase protection assay. The hESRL1a mRNA is differentially expressed in various human tissues. The nucleotide sequence adjacent to the transcription start sites of the ESRL1 lacks the typical TATA and CAAT boxes but is GC rich and contains 10 consensus Sp1-binding elements and two E boxes. The region that contains these transcription factor-binding elements showed a high level of promoter activity when transiently transfected into RL95-2 cells.

Blotting, Northern↗
PubMed1992

cDNA cloning and sequencing of rat monoamine oxidase A: comparison with the human and bovine enzymes.

1. The nucleotide and deduced amino acid sequences of rat liver MAO A were determined, and sequence identities among MAO A and B from rat, human and bovine were compared. 2. MAO A from rat exhibited greater than 85% sequence identity with bovine and human MAO A, and 70% identity with rat MAO B. 3. Rat adrenal cDNAs were restriction mapped, partially sequenced and found to be identical to rat liver MAO A, suggesting that these two tissues express the same polypeptide.

Adrenal Medulla↗
PubMed1988

Nucleotide and deduced amino acid sequence of the E1 alpha subunit of human liver branched-chain alpha-ketoacid dehydrogenase.

A 1552-bp cDNA for the E1 alpha subunit of branched-chain alpha-ketoacid dehydrogenase (BCKDH) was isolated from a human liver cDNA library. The cDNA contained a 1134-bp open reading frame that encoded 378 amino acid (aa) residues of the enzyme and 418 bp of 3'-untranslated sequence. The deduced amino acid sequence of the human protein shows 96% identity with that of the rat enzyme subunit. Those 117-aa residues surrounding the phosphorylation sites are completely conserved between man and rat. BCKDH E1 alpha showed considerable amino acid sequence similarity with pyruvate dehydrogenase E1 alpha, particularly in the region of the two principal phosphorylation sites of these proteins. Northern blots of human liver and skin fibroblasts demonstrated a single 1.8-kb mRNA band, with a higher level of E1 alpha mRNA in liver than in normal fibroblasts. Fibroblasts from a patient with thiamine-responsive maple syrup urine disease (MSUD) contained an mRNA of the same size and abundance as that of normal fibroblasts. Genomic DNA from normal and MSUD fibroblasts gave the same restriction maps on Southern blots, and the gene was approximately 10-kb in size.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1990

Molecular and phenotypic variation in the achaete-scute region of Drosophila melanogaster.

Variation in quantitative characters underlies much adaptive evolution and provides the basis for selective improvement of domestic species, yet the genetic nature of quantitative variation is poorly understood. Many loci affecting quantitative traits have been identified by the segregation of mutant alleles with major qualitative effects. These alleles may represent an extreme of a continuum of allelic effects, and most quantitative variation could result from the segregation of alleles with subtle effects at loci identified by alleles with major effects. The achaete-scute complex in Drosophila melanogaster plays a central part in bristle development and has been characterized at the molecular level. The hypothesis that naturally occurring quantitative variation in bristle number could be associated with wild-type alleles of achaete-scute was tested by correlating phenotypic variation in bristle number with molecular variation in restriction maps in this region among chromosomes extracted from natural populations. DNA insertion variation in the achaete-scute region was found to be strongly associated with variation in bristle number.

Alleles↗
PubMed1989

Multipartite structure of mitochondrial DNA in a fungal longlife mutant.

Rearrangements in the mitochondrial genome of the longlife mutant ex1 of the ascomycete Podospora anserina have led to a heterogeneous population of subgenomic molecules. The restriction maps of individual subcircles were established using overlapping recombinant lambda phages isolated from an ex 1 mtDNA/EMBL3 library. The formation of the subcircles and the resulting multipartite organization of the ex1 mtDNA are discussed.

Ascomycota↗
PubMed1990

Isolation and restriction endonuclease analysis of a tetracycline resistance plasmid from Staphylococcus hyicus.

A plasmid of 4.550 kb, conferring resistance to tetracycline, was demonstrated in Staphylococcus hyicus cultures from piglets with exudative epidermidis. The plasmid-encoded properties were determined both by curing and interspecific protoplast transformation experiments. The tetracycline resistance (TET) plasmid, designated pST1, was characterized by restriction endonuclease analysis and a preliminary restriction map was constructed. The pST1 plasmid was demonstrated in 19 (57.6%) of 33 S. hyicus cultures by Southern blot hybridization. It was also detectable by electron microscopy.

Animals↗
PubMed1990

[Isolation and characteristics of mitochondrial DNA from Acremonium chrysogenum].

Circular mDNAs 26.85 and 26.94 kb in length were isolated from two isogenic strains of A. chrysogenum producing cephalosporin C. The strains differed in antibiotic production capacity. Restriction analysis of the mDNAs was performed with using 6 endonucleases. Comparison of the restriction data revealed identity of mDNAs. A restriction map of the mDNAs was constructed. It is useful as a basis for further studies with molecular cloning.

Acremonium↗
PubMed1992

Characterization of yeast artificial chromosomes containing interleukin genes on human chromosome 5.

To understand better the organization and linkage of the interleukin genes, IL4 and IL5, we prepared long-range restriction maps of five yeast artificial chromosomes (YACs) containing IL5. We determined that IL4 and IL5 are within 100-170 kb, and that the regions surrounding these genes contain several GC-rich areas. Fluorescence in situ chromosomal analysis demonstrated that three of the five YAC clones contain non-contiguous genomic sequences originating from multiple human chromosomes.

Base Sequence↗
PubMed1994

The 9-kDa calbindin gene of Rousettus aegyptiacus: its identification and isolation from a genomic library.

A genomic library of the fruit bat (Rousettus aegyptiacus) was constructed in lambda phage gt11. The titre of the library was determined to be 2 x 10(5) pfu/ml. The genomic library was amplified and the titre of the amplified library increased 300-fold to 7 x 10(7) pfu/ml. The library was screened by in situ hybridization techniques using a fragment of the mouse 9-kDa calbindin cDNA as a probe. Screening of 10(5) plaques yielded a positive clone. Three additional rounds of screening were performed to purify the positive. Lambda phage DNA was isolated from the positive clone and restriction digest analysis, followed by hybridization studies, was performed on these digests in order to determine the location of the bat 9-kDa calbindin gene in the insert of the lambda phage vector. Restriction maps so derived were interpreted from the published sequence for the rat 9-kDa calbindin gene and indicate the successful isolation of the 9-kDa calbindin gene of Rousettus aegyptiacus.

Animals↗
PubMed1989

Cloning and characterization of variable-sized gypsy mobile elements in Drosophila melanogaster.

A cosmid genomic library from a known gypsy-induced forked mutation, f1, was screened by 32P-labeled gypsy transposable element. Of more than 250 positive clones we randomly selected 21 for in situ hybridization to wild-type polytene chromosomes. Two clones hybridized to region 15F on the X-chromosome, the cytological position of forked. A third clone hybridized to at least 17 sites on the chromosomes indicating the presence of repetitive sequences in the gypsy flanking DNA. All clones labeled the centromeric regions heavily. Ten clones, including the two hybridizing at 15F, were chosen for further analysis, and restriction mapping allowed us to place them into three groups: (1) full-length, (2) slightly diverging, and (3) highly diverging gypsy elements. Group (2) is missing the XbaI site in both their long terminal repeats (LTRs) as well as the middle HindIII site; four of these gypsy elements also have a approximately 100-bp deletion at the 5' LTR. The group (3) gypsy transposons are missing one LTR and also have highly diverging DNA sequences. The restriction analyses further imply that most of these different gypsy elements are present in more than one copy in the genome of the f1 stock used in this study. The results raise intriguing questions regarding the significance of transposable elements in evolution and biological functions.

Animals↗
PubMed1990

DNA sequence of the purC gene encoding 5'-phosphoribosyl-5-aminoimidazole-4-N-succinocarboxamide synthetase and organization of the dapA-purC region of Escherichia coli K-12.

5'-Phosphoribosyl-5-aminoimidazole-4-N-succinocarboxamide synthetase (EC 6.3.2.6), encoded by the purC gene of Escherichia coli K-12, catalyzes the synthesis of 5'-phosphoribosyl-5-aminoimidazole-4-N-succinocarboxamide from 5'-phosphoribosyl-5-aminoimidazole-4-carboxylic acid. The mature protein, as deduced from the purC structural gene sequence, contains 237 amino acids and has a calculated Mr of 26,998. The control region of the purC gene was identified by primer extension mapping of the 5' end of the purC mRNA. The purC control region contains a binding site for and is regulated by the purine repressor, the product of the purR gene. An unusual feature of the 5' untranslated region of the purC mRNA is the presence of a repetitive extragenic palindrome sequence normally found in intercistronic or 3' untranslated regions. The DNA sequence was extended 1.281 kilobases upstream of the purC structural gene and overlapped with the previously determined dapA sequence. Termination of transcription from the dapA-purC intercistronic region may occur within the -35 region of the purC control region. The purC gene has been positioned on the E. coli restriction map and is transcribed in a counterclockwise direction.

Amino Acid Sequence↗
PubMed1991

Physical linkage and transcriptional orientation of the tdc operon on the Escherichia coli chromosome.

The physical and genetic structure of 37 kilobases of DNA encompassing the tdc region at 68.3 min of the Escherichia coli chromosome was determined by DNA sequence analysis and restriction mapping. Re-examination of new data concerning the direction of transcription of the tdc operon revealed that in strain W3110 the tdc region is located on a transposable segment of DNA.

Cloning, Molecular↗
PubMed2000

Evolutionary conservation of the apolipoprotein E-C1-C2 gene cluster on bovine chromosome 18q24.

We have constructed a long-range restriction map spanning about 250 kb on bovine chromosome 18q24. Our results show that the apolipoprotein C2 (APOC2) gene is located about 25 kb from the APOE gene. Four putative CpG islands are also indicated in the map. Interestingly, a minisatellite located in the third intron of the human and mouse APOC2 genes was also found at identical position in the bovine gene and revealed high sequence identity comparing with the two corresponding sequences. By means of cosmid mapping, we further demonstrate that the APOE-APOC1-APOC2 gene cluster is evolutionary conserved in cattle.

Animals↗
PubMed1992

Isolation and structural analysis of a 1.2-megabase N-myc amplicon from a human neuroblastoma.

Oncogene amplification is observed frequently in human cancers, but little is known about the mechanism of gene amplification or the structure of amplified DNA in tumor cells. We have studied the N-myc amplified domain from a representative neuroblastoma cell line, SMS-KAN, and compared the map of the amplicon in this cell line with that seen in normal DNA. The SMS-KAN cell line DNA was cloned into yeast artificial chromosomes (YACs), and clones were identified by screening the YAC library with amplified DNA probes that were obtained previously (B. Zehnbauer, D. Small, G. M. Brodeur, R. Seeger, and B. Vogelstein, Mol. Cell. Biol. 8:522-530, 1988). In addition, YAC clones corresponding to the normal N-myc locus on chromosome 2 were obtained by screening two normal human YAC libraries with these probes, and the restriction maps of the two sets of overlapping YACs were compared. Our results suggest that the amplified domain in this cell line is a approximately 1.2-Mb circular molecule with a head-to-tail configuration, and the physical map of the normal N-myc locus generally is conserved in the amplicon. These results provide a physical map of the amplified domain of a neuroblastoma cell line that has de novo amplification of an oncogene. The head-to-tail organization, the general conservation of the normal physical map in the amplicon, and the extrachromosomal location of the amplified DNA are most consistent with the episome formation-plus-segregation mechanism of gene amplification in these tumors.

Base Sequence↗
PubMed2002

A bacteriophage reagent for Salmonella: molecular studies on Felix 01.

Felix 01 (F01) is a bacteriophage originally isolated by Felix and Callow which lyses almost all Salmonella strains and has been widely used as a diagnostic test for this genus. Molecular information about this phage is entirely lacking. In the present study, the DNA of the phage was found to be a double-stranded linear molecule of about 80 kb. 11.5 kb has been sequenced and in this region A + T content is 60%. There are relatively few restriction endonuclease cleavage sites in the native genome and clones show this is due to their absence rather than modification. A restriction map of the genome has been constructed. The ends of the molecule cannot be ligated although they contain 5' phosphates. At least 60% of the genome must encode proteins. In the sequenced portion, many open reading frames exist and these are tightly packed together. These have been examined for homology to published proteins but only 1 to 17 shows similarity to known proteins. F01 is therefore the prototype of a new phage family. On the basis of restriction sites, codon usage and the distribution of nonsense codons in the unused reading frames, a strong case can be made for natural selection that reacts to mRNA structure and function.

Base Sequence↗