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PubMed1989

Isolation and characterization of the complete human beta-myosin heavy chain gene.

The entire gene coding for the human beta-myosin heavy chain has been isolated from genomic EMBL3A phage libraries by chromosomal walking starting from clone gMHC-1, reported earlier (Appelhans and Vosberg 1983). gMHC-1 has been shown to carry coding information for the C-terminal two-thirds of beta-myosin heavy chain, which is expressed in cardiac muscle and in slow skeletal muscle fibers (Lichter et al. 1986). Three DNA clones were identified as overlapping with gMHC-1 by restriction mapping and DNA sequencing. They span a 30-kb region in the genome. About 22 kb extend from the initiation codon ATG to the poly(A) addition site. The clones include about 4 kb of 5' flanking sequences upstream of the promoter. Comparisons of beta- and alpha-myosin heavy chain sequences indicate that gene duplication of the cardiac myosin heavy chain isogenes preceded the mammalian species differentiation.

Animals↗
PubMed1990

Sequence of the precursor of bovine liver mitochondrial aldehyde dehydrogenase as determined from its cDNA, its gene, and its functionality.

The partial sequence coding for bovine liver mitochondrial aldehyde dehydrogenase was previously reported (Farres, J., Guan, K.L., and Weiner, H., 1989, Eur. J. Biochem. 180, 67-74); cDNA coding for the N-terminal region has now been obtained by primer extension. The deduced 520 amino acids contained 499 residues corresponding to the mature mitochondrial aldehyde dehydrogenase which was one residue shorter than other mammalian mitochondrial aldehyde dehydrogenases. The N-terminal signal peptide had only 21 amino acids but showed high sequence identity to the signal sequences of human and rat enzymes, which have 17 and 19 amino acids, respectively, and had general characteristic features of typical mitochondrial signal peptides. Although the direct protein sequence of the signal peptide was not available, a mRNA coding for a 57-kDa precursor was proven to exist. The bovine signal peptide was able to direct the import of bovine aldehyde dehydrogenase precursor into isolated liver mitochondria. The complete nuclear gene coding for bovine mitochondrial aldehyde dehydrogenase was found to span at least 25 kb. A restriction map of this gene was constructed. Two potential transcription start sites were identified by primer extension and S1 nuclease protection. Several SpI transcription factor recognition sequences were found in the 5'-region of the gene. TATA box and CAAT box like sequences were found to be exist 18 and 64 bp upstream from the major transcription start site, respectively.

Aldehyde Dehydrogenase↗
PubMed1990

Cloning and sequencing of the gene encoding cytochrome c-551 from Pseudomonas aeruginosa.

The cytochrome c-551 gene from Pseudomonas aeruginosa was cloned by using two oligonucleotide probes, which had been synthesized based on the known primary structure of the protein. The restriction map of the cloned DNA and sequence analysis showed that the cytochrome c-551 gene is located 50 bp downstream of the nitrite reductase gene, which has recently been cloned and sequenced. DNA sequence analysis also indicated that cytochrome c-551 is synthesized in vivo as a precursor having an amino-terminal signal sequence consisting of 22 amino acid residues.

Amino Acid Sequence↗
PubMed1999

Molecular cloning and characterization of two sets of alpha-theta genes in the rat alpha-like globin gene cluster.

The highly heterogeneous rat hemoglobin system was investigated at the gene level. Two regions of the alpha-like globin gene cluster from a Wistar rat were isolated. Four lambda Dash recombinant clones carrying rat alpha-like globin genes were localized on two distinct gene regions. A region of approximately 16kb was found to contain the 5'-IIalpha1-psi theta 1-3' loci, and another of approximately 24kb the 5'-IIalpha2-psi theta2-psiI alpha3-3' loci. Both IIalpha1 and IIalpha2 are considered to be active, coding the IIalpha-globin chain. The nt sequences of IIalpha1 and IIalpha2 are identical except for six nt in the non-coding region. The psiI alpha3 locus is a truncated pseudogene. The putative promoter region of an alpha-like globin gene is joined directly to the third exon, homologous to that of Ialpha-globin cDNA. psi theta1 and psi theta2 are also pseudogenes, as evidenced by several deletions located in the protein-coding regions of these loci. The psi theta1 and psi theta2 loci exhibit extensive homology, but the restriction maps of these genes and their flanking regions differ considerably. Genomic Southern blot analyses of the total liver DNA from six rats showed the existence of three theta-globin-related genes, including psi theta1 and psi theta2. These results indicate that the two gene regions investigated are not allelic variants, but may be generated by block duplication. This is the first report of the existence of rodent theta-globin genes.

Animals↗
PubMed1994

A physical comparison of chromosome III in six strains of Saccharomyces cerevisiae.

We have tested the clones used in the European Yeast Chromosome III Sequencing Programme for possible artefacts that might have been introduced during cloning or passage through Escherichia coli. Southern analysis was performed to compare the BamHI, EcoRI, HindIII and PstI restriction pattern for each clone with that of the corresponding locus on chromosome III in the parental yeast strain. In addition, further enzymes were used to compare the restriction maps of most clones with the map predicted by the nucleotide sequence (Oliver et al., 1992). Only four of 506 6-bp restriction sites predicted by the sequence were not observed experimentally. No significant cloning artefacts appear to disrupt the published sequence of chromosome III. The restriction patterns of six yeast strains have also been compared. In addition to two previously identified sites of Ty integration on chromosome III (Warmington et al., 1986; Stucka et al., 1989; Newlon et al., 1991), a new polymorphic site involving Ty retrotransposition (the Far Right-Arm transposition Hot-Spot, FRAHS) has been identified close to CRY1. On the basis of simple restriction polymorphisms, the strains S288C, AB972 and W303-1b are closely related, while XJ24-24a and J178 are more distant relatives of S288C. A polyploid distillery yeast is heterozygous for many polymorphisms, particularly on the right arm of the chromosome.

Artifacts↗
PubMed1994

Construction and features of lambda EMBL3cosW, a lambda replacement vector for detailed analysis of large regions of genomic DNA.

A phage lambda replacement vector, lambda EMBL3cosW, is described which expedites detailed analysis of large regions of chromosomal DNA. Two features of the vector aid this process. Firstly, the replaceable stuffer in lambda EMBL3cosW is flanked by SP6 and T7 promoters so that end-specific hybridisation probes can be rapidly generated from cloned inserts for identification of sequentially overlapping clones in genomic libraries. Secondly, because all the phage coding sequences in the vector (which are placed to the right of the replaceable stuffer) can be removed from cloned inserts by cleavage with NotI, restriction mapping of cloned inserts using partial digest strategies is greatly facilitated. Other features of the vector are: (1) strategically placed BamHI and XhoI sites for the cloning of genomic DNA partially digested with MboI or Sau3AI by two different methods; (2) SalI and SfiI sites for the isolation of intact cloned inserts; and (3) transcription terminators to insulate vector genes from transcriptional interference from cloned insert DNAs.

Bacteriophage lambda↗
PubMed1989

ExoMeth sequencing of DNA: eliminating the need for subcloning and oligonucleotide primers.

A method is reported for sequencing DNA based on exonuclease III digestion and strand protection by using modified nucleoside triphosphates. Up to 10 kilobases of sequence information may be obtained from each strand of a given template without subcloning. Prior knowledge of the restriction map is not important; prior knowledge of any of the sequence is not required. Nor are oligonucleotide primers needed. Double-stranded cosmids, plasmids, lambda phage, or linear molecules (including amplified molecules) may be used as starting material. The method creates a single-stranded template from these starting molecules, thus generating high-quality sequence ladders. Most commonly used DNA polymerases may be utilized, including reverse transcriptase and T7 DNA polymerase. The approach is "ordered", so little time is wasted on redundant sequencing.

Base Sequence↗
PubMed1989

The abnormal oocyte phenotype is correlated with the presence of blood transposon in Drosophila melanogaster.

The abnormal oocyte mutation (2;44) originates in the wild: it confers no visible phenotype on homozygous abo males or females, but homozygous abo females produce defective eggs and the probability of their developing into adults is much lower than that of heterozygous sister females. We isolated by chromosome walking 200 kb of DNA from region 32. This paper reports that a restriction enzyme site polymorphism analysis in wild type and mutant stocks allowed us to identify a DNA rearrangement present only in stocks carrying the abo mutation. The rearrangement is caused by a DNA insert on the abo chromosome in region 32E which, by restriction map and sequence analysis, was identified as copia-like blood transposon. The transposon, in strains that had remained in abo homozygous conditions for several generations and had lost the abo maternal-effect, was no longer present in region 32E. Certain features of the abo mutation, discussed in the light of this finding, may be ascribed to the nature of the particular allele studied.

Animals↗
PubMed1997

Various fructose-1,6-bisphosphatase mRNAs in mouse brain, liver, kidney and heart.

The mouse fructose-1,6-bisphosphatase (FBPase) cDNA was previously cloned from testicular teratocarcinoma cultured cells (F9 cells). Using this published nucleotide sequence four primer sets were defined and used to amplify FBPase transcript from cerebral cortex, heart, kidney, liver and testis of male C57B1/6 mice. Only one primer set was efficient in all total RNA prepared from the various tissues. The restriction maps of these RNA amplification products suggested the existence of three different FBPase transcripts; this was confirmed by the nucleotide sequences of the FBPase transcripts and by the deduced amino acid sequences. These data are consistent with the existence of three different FBPase genes. This may be relevant in neurological disease in which abnormalities of brain glucose metabolism are involved.

Amino Acid Sequence↗
PubMed1988

Characterization of bacteriophage phi C69 of Saccharopolyspora erythraea and demonstration of heterologous actinophage propagation by transfection of Streptomyces and Saccharopolyspora.

A bacteriophage, designated phi C69, isolated from a culture of Saccharopolyspora erythraea was characterized. The phage propagates on Sac. erythraea NRRL 2338 but does not infect 10 Streptomyces or 3 Micromonospora species tested. It infects Sac. erythraea NRRL 2359 but does not produce infectious phage particles in this host. phi C69 is approximately 40 kb in length and contains cohesive ends. A cos fragment containing ligated phage DNA ends was cloned in Escherichia coli. Restriction maps of the phage DNA and the cos fragment for several enzymes are shown. Transfection of both Sac. erythraea and Streptomyces lividans with phi C69 resulted in approximately equal titres of infectious phage particles produced from approximately the same number of regenerating cells. Transfection of Sac. erythraea with DNA from Streptomyces phages SH10 and KC404 also resulted in the production of infectious phage particles. The basis for differences among hosts in susceptibility to infection by various actinophages is discussed.

Bacteriophages↗
PubMed1993

Rapid identification of overlapping YACs in the MEN2 region of human chromosome 10 by hybridization with Alu element-mediated PCR products.

An overlapping set of 21 yeast artificial chromosomes (YACs) spanning the RET proto-oncogene [Takahashi et al., Oncogene 3 (1988) 571-578] and D10S102 markers on human chromosome 10 was isolated in a series of hybridization-based chromosomal walks in a YAC library. Genetic linkage analyses implicate this chromosomal region as the location of the gene (MEN2A) responsible for multiple endocrine neoplasia type 2A. Four YACs carrying a RET sequence-tagged site (STS) and two YACs carrying a D10S102 STS were used to initiate chromosome walks. These were based on hybridization of Alu element-mediated polymerase chain reaction (Alu-PCR) products from YACs to dot blots of Alu-PCR products from complex pools of YAC clones. The hybridization anchor content of YACs identified in the walks was confirmed by probing blots of Alu-PCR products from individual YACs and by comparing Alu-PCR fingerprints of each YAC. Ten hybridization-based Alu-PCR anchors and three STS anchors were ordered within eleven intervals created by the 21 overlapping YACs. The order of anchors requiring the fewest gaps in the YACs is consistent with the walking results and establishes the STS anchor order as D10S102-D10S94-RET. The overlapping set of YACs represents about 1.55 Mb of the human genome according to restriction mapping of four representative YACs in the contig. These results demonstrate the power of Alu-PCR hybridization for chromosomal walking and provide a rich source of overlapping YACs which can be used to identify candidate MEN2A genes.

Base Sequence↗
PubMed1991

Conservation in structure of TOC1 transposons from Chlamydomonas reinhardtii.

TOC1 transposons from Chlamydomonas reinhardtii have an unusual arrangement of long terminal repeats. Polymorphic regions between TOC1 transposons were identified by restriction mapping on Southern blots. The variation in size of an internal MluI fragment defines two subclasses of TOC1 elements. Full-length cloned members of each subclass of TOC1 element were compared by electron microscope heteroduplex analysis. The cloned elements were co-linear over their entire length with no large sequence discontinuities. Base substitutions and small insertion/deletion events of less than 50 bp are responsible for forming the two subclasses of TOC1 elements.

Animals↗
PubMed1989

The essential nature of teichoic acids in Bacillus subtilis as revealed by insertional mutagenesis.

A 30 kb DNA segment from the region of the Bacillus subtilis strain 168 chromosome which contains most, if not all, loci specifically involved in teichoic acid biosynthesis, has been cloned. A restriction map was established to which genetic markers were assigned. Four loci, tagA, tagB, gtaA and gtaD, are located on a DNA segment of about 7 kb, whereas the gtaB locus lies some 10 kb distant. The tagA and tagB loci are apparently transcribed independently. Insertional mutagenesis, using integrational plasmids carrying relevant fragments from the tag region, provides strong evidence that biosynthesis of polyglycerol phosphate [poly(groP)], so far largely considered as a dispensable polymer, is in fact essential for growth.

Bacillus subtilis↗
PubMed1989

Addition of cloned beta-glucosidase enhances the degradation of crystalline cellulose by the Clostridium thermocellum cellulose complex.

A thermostable beta-glucosidase from Clostridium thermocellum which is expressed in Escherichia coli was used to determine the substrate specificity of the enzyme. A restriction map of the beta-glucosidase gene cloned in plasmid pALD7 was determined. Addition of the E. coli cell extract (containing the beta-glucosidase) to the cellulase complex from C. thermocellum increased the conversion of crystalline cellulose (Avicel) to glucose. The increase was specifically due to hydrolysis of the accumulated cellobiose. A cellulose degradation process using beta-glucosidase to assist the potent cellulase complex of C. thermocellum, as shown here can open the way for industrial saccharification of cellulose to glucose.

Cellulase↗
PubMed1989

Characterization of a gram-positive broad-host-range plasmid isolated from Lactobacillus hilgardii.

Two plasmids, pLAB1000 and pLAB2000 (3.3 and 9.1 kb, respectively), have been isolated from a grass silage strain of Lactobacillus hilgardii. Both plasmids were cloned in Escherichia coli and characterized through restriction mapping. A 1.6-kb XbaI-SacI fragment of pLAB1000 appeared to be sufficient for autonomous replication in Lactobacillus plantarum and in Bacillus subtilis. Different shuttle vectors for E. coli and gram-positive bacteria were developed using the pLAB1000 plasmid. These could stably be maintained in Lactobacillus, Enterococcus, and Bacillus under selective conditions. Plasmids sharing DNA homologies with pLAB1000 have been observed in different strains of the related species L. plantarum.

DNA, Bacterial↗
PubMed1989

Cloning and characterization of the 5' end (exon 1) of the gene encoding human factor X.

Human blood-coagulation factor X (hBCFX) is a serine protease zymogen which participates in the middle phase of blood coagulation. Recently, we and others have reported the cDNA sequences. At present, partial hBCFX gene structure is available. In this paper, we report the isolation of two genomic clones, the X-emb lambda phage clone encoding exons 1 and 2 of the hBCFX gene, and the X-cos cosmid clone encoding exons 2-8. The restriction map of the hBCFX region spans 55 kb. The gene itself was found to be 27 kb long. The sequence of the 5' region of the hBCFX gene has been determined and reveals an ATTTG pentanucleotide, which also occurs in a similar location in the genes encoding factors VII, IX, protein CC and C prothrombin, suggesting that this motif might be of importance in the regulation of these genes.

Base Sequence↗
PubMed1995

Molecular organization of the gene encoding murine transforming growth factor beta 1.

A detailed restriction map of the murine TGF-beta 1 locus (encoding transforming growth factor beta 1) was established and the precise junctional sequences of its seven exons and corresponding introns were elucidated. While the exons ranged in size from 78 to 357 bp, the introns ranged from about 1 to 6.5 kb. The promoter-proximal segment contains many putative regulatory motifs which may dictate TGF-beta 1 gene transcription in response to varied pathophysiological stimuli.

Animals↗
PubMed1996

Isolation and characterization of the versicolorin B synthase gene from Aspergillus parasiticus. Expansion of the aflatoxin b1 biosynthetic gene cluster.

Versicolorin B synthase catalyzes the side chain cyclization of racemic versiconal hemiacetal to the bisfuran ring system of(-)-versicolorin B, an essential transformation in the aflatoxin biosynthetic pathway of Aspergillus parasiticus. The dihydrobisfuran is key to the mutagenic nature of aflatoxin B1. The protein, which shows 58% similarity and 38% identity with glucose oxidase from Aspergillus niger, possesses an amino-terminal sequence homologous to the ADP-binding region of other flavoenzymes. However, this enzyme does not require flavin or nicotinamide cofactors for its cyclase activity. The 643-amino acid native enzyme contains three potential sites for N-linked glycosylation, Asn-Xaa-Thr or Asn-Xaa-Ser. The cDNA and genomic clones of versicolorin B synthase were isolated by screening the respective libraries with random-primed DNA probes generated from an exact copy of an internal vbs sequence. This probe was created through polymerase chain reaction by using nondegenerate polymerase chain reaction primers derived from the amino acid sequences of peptide fragments of the enzyme. The 1985-base genomic vbs DNA sequence is interrupted by one intron of 53 nucleotides. Southern blotting, nucleotide sequencing, and detailed restriction mapping of the vbs-containing genomic clones revealed the presence of omtA, a methyltransferase active in the biosynthesis, 3.3 kilobases upstream of vbs and oriented in the opposite direction from vbs. The presence of omtA in close proximity to vbs supports the theory that the genes encoding the aflatoxin biosynthetic enzymes in A. parasiticus are clustered.

Aflatoxin B1↗