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PubMed1992

Molecular cloning of a determinant coding for fimbrial antigen F165(1), a Prs-like fimbrial antigen from porcine septicaemic Escherichia coli.

The genetic determinant coding for F165(1) fimbriae was cloned from the chromosome of the porcine Escherichia coli wild-type strain 4787 (O115:K-:H51:F165). The fimbrial determinant was further subcloned into the BamHI site of pACYC184 and a restriction map was established. On Southern hybridization, identity between the chromosomally encoded prs-like determinant of strain 4787 and its cloned counterparts was demonstrated. The cloned F165(1) fimbriae and those of the wild-type strain possessed a major protein subunit of molecular mass 18.5 kDa. Strains expressing F165(1) fimbriae were detected using an F165-specific polyclonal antiserum and caused mannose-resistant haemagglutination and agglutination of Forssman latex beads. Antiserum against the cloned F165(1) fimbriae recognized a 18.5 kDa band in the parent strain 4787.

Amino Acid Sequence↗
PubMed1998

Study on the genetic determinants responsible for expression and assembly of CS3 fimbriae.

Colonization factor antigens (CFAs) are important virulent factors and protective antigens of enterotoxigenic Escherichia coli (ETEC). Among the known CFAs of human origin, CS3 is a major fimbriae antigen. To study the genes associated with the expression and assembly of CS3 is a key step to delineate the regulating mechanism of expression and assembly of the fimbriae. In this paper we analyzed the restriction map of the CS3 genetic determinant and successfully cloned the CS3 subunit gene and the region encoding the auxiliary proteins. Expression results of the recombinant plasmids in minicells showed that the genetic determinant of CS3 fimbriae encodes six polypeptides with molecular weights 15, 17, 24, 27, 48, and 90 kDa. Western blotting and complementary expression analysis among the different mutants showed that the 15 kDa/17 kDa proteins compose the CS3 subunit and the 17 kDa protein is the precursor. The others may be necessary for the assembly of CS3 fimbriae. The relative locations of various genes of the CS3 genetic determinant were also determined.

Bacterial Proteins↗
PubMed1993

Organization and evolution of an alpha satellite DNA subset shared by human chromosomes 13 and 21.

The structure of the alpha satellite DNA higher-order repeat (HOR) unit from a subset shared by human chromosomes 13 and 21 (D13Z1 and D21Z1) has been examined in detail. By using a panel of hybrids possessing either a chromosome 13 or a chromosome 21, different HOR unit genotypes on chromosomes 13 and 21 have been distinguished. We have also determined the basis for a variant HOR unit structure found on approximately 8% of chromosomes 13 but not at all on chromosomes 21. Genomic restriction maps of the HOR units found on the two chromosome 13 genotypes and on the chromosome 21 genotype are constructed and compared. The nucleotide sequence of a predominant 1.9-kilobasepair HOR unit from the D13Z1/D21Z1 subset has been determined. The DNA sequences of different alpha satellite monomers comprising the HOR are compared, and the data are used to develop a model, based on unequal crossing-over, for the evolution of the current HOR unit found at the centromeres of both these chromosomes.

Animals↗
PubMed1988

Molecular cloning and characterization of human ret-II oncogene.

A transformant having ret-II was obtained by transfection of NIH3T3 cells with DNA from a human sigmoid colon cancer. Comparison of the restriction map of ret-II with that of proto-ret revealed that the ret-II has rearrangements both 5' and 3' to the sequence coding for the kinase domain. However, only the upstream rearrangement was consistently observed in all transformants, suggesting that the upstream rearrangement resulted in the activation of proto-ret. The recombination point and replaced sequence of ret-II were different from those previously reported for ret (Takahashi et al., 1985). In addition, ret-II does not retain region coding for transmembrane of proto-ret. This rearrangement, however, was not detected in the original tumor DNA by Southern blot analysis.

Animals↗
PubMed1996

Amplification, cloning, and sequence comparison of the growth hormone gene for carp (Cyprinus carpio) by the polymerase chain reaction.

Total RNA was isolated from carp pituitary gland. The first strand cDNA was synthesized using oligo(dT) 12-18 as a primer, the total RNA as a template, and AMV reverse transcriptase. Next the polymerase chain reaction (PCR) was performed using the first strand cDNA as a template, the synthetic 29 oligonucleotides as primer and Taq DNA polymerase (94 degrees C, 60 s; 55 degrees C, 30 s; 72 degrees C, 50 s; 35 cycles). After PCR amplification, the products were cloned into an E. coli expression vector (PBluescript II KS+/-). The result of the sequence analysis and the restriction map shows that an open reading frame of the carp growth hormone gene contains 630 base pairs which code for a polypeptide of 210 amino acids including 22 amino acids of the signal peptide and 188 amino acids of the nature growth hormone. Nucleotide sequence and amino acid sequence of our carp growth hormone gene are the same as Koren's carp GH cDNA in the coded region. Compared with Chao's carp GH cDNA, the homology of nucleotide sequence and amino acid sequence for our carp growth hormone gene is 95.6% and 96.7%, respectively, in the coded region.

Amino Acid Sequence↗
PubMed1994

A kalilo-like linear plasmid in Louisiana field isolates of the pseudohomothallic fungus Neurospora tetrasperma.

Two Louisiana strains of Neurospora tetrasperma contain a linear plasmid (LA-kalDNA) with a restriction map identical to the Hawaiian Neurospora intermedia senescence plasmid, kalDNA, but with termini 100 nucleotide pairs shorter. One of these strains also bore a circular plasmid similar to the Hawaiian circular plasmid Hanalei-2. One species probably acquired both plasmids from the other by horizontal transfer, at a time sufficiently distant for sequence divergence to take place. Many LA-kalDNA-bearing derivative strains senesced, but this plasmid does not guarantee senescence. Furthermore, LA-kalDNA does not insert into mtDNA. One senescent strain showed no LA-kalDNA. The plasmids are effectively transmitted via the pseudohomothallic sexual cycle. Single mating-type derivatives transmit plasmids maternally.

Biological Evolution↗
PubMed1994

Structure of a gene coding for human dihydrodiol dehydrogenase/bile acid-binding protein.

The structure and sequence of the gene (DD/BABP) encoding a human dihydrodiol dehydrogenase/bile acid-binding protein (DD/BABP) were determined by analysis of genomic clones. Several overlapping clones containing parts of the gene were isolated from a lambda EMBL3 SP6/T7 library and characterized by restriction mapping and DNA sequencing. The gene spans approx. 16kb and consists of nine exons. The sizes of the exons range from 77 to 167 bp, whereas the intron sizes range from 375 to 3430 bp. The transcription start point (tsp) is located at -56 bp of the first ATG codon, as determined by primer extension. Neither a TATA box nor a CAT box was found upstream from the tsp. Southern blot analysis of the human genomic DNA, using the cDNA as the probe, revealed several additional hybridizing DNA bands, suggesting the existence of multiple related genes.

Amino Acid Sequence↗
PubMed1991

Molecular characterization of a cluster of at least two glucosyltransferase genes in Streptococcus salivarius ATCC 25975.

The oral micro-organism Streptococcus salivarius ATCC 25975 synthesizes extracellular glucosyltransferases (GTFs) which polymerize the glucose moiety of sucrose into glucan polymers. Two separate genes encoding the activities of a GTF-I (a GTF that synthesizes an insoluble product) and a GTF-S (a GTF that synthesizes soluble product) were cloned into bacteriophage lambda L47.1. The inserts in the lambda-clones were characterized by restriction mapping and Southern hybridization and were found to overlap, implying that the two genes lay very close to one another on the S. salivarius chromosome. Both genes were subcloned into phagemid vector pIBI30 where they were expressed at a high level. The GTF-I-encoding gene was named gtfJ and the GTF-S-encoding gene, gtfK. Nucleotide sequencing showed that gtfJ and most probably gtfK were closely related to the gtf genes of the mutans streptococci. Sequence alignment also indicated that gtfK lay very close to and downstream from gtfJ, and that both were transcribed in the same direction.

Amino Acid Sequence↗
PubMed2000

Identification of a DNA region from lactococcal phage sk1 protecting phage 712 from the abortive infection mechanism AbiF.

Bacteriophage 712 is a small isometric-headed phage which is sensitive to the lactococcal abortive infection mechanism AbiF. Its 29.6-kb DNA genome was characterized by restriction mapping and transcriptional analysis. Construction of a gene bank of lactococcal phage sk1, which is insensitive to the action of AbiF, in Lactococcus lactis containing AbiF resulted in the identification of a 324-bp DNA fragment which reduced the effect of the abortive infection mechanism on phage 712. Analysis of this region provided evidence that the action of AbiF is related to the cos ends of small isometric-headed phages. Sequence analysis of a 3.2-kb segment containing the middle operon and the cos ends of phage 712 genome allowed comparison of this part of the phage 712 genome with the equivalent sequences of four other small isometric-headed phages.

Cloning, Molecular↗
PubMed1998

Fine exon-intron structure of the Fanconi anemia group A (FAA) gene and characterization of two genomic deletions.

Fanconi anemia (FA) is a genetically heterogeneous disease with at least eight genes on the basis of complementation groups (FAA to FAH). The analysis of the FAA gene in patients suggested the existence of deletions, none of which have thus far been characterized at the genomic level. A detailed restriction map of the FAA gene with the fine localization of its 43 exons is reported in this paper. We also describe the first two genomic deletions, one of 5.0 kb and another of at least 120 kb. The former was likely the result of a recombination between related Alu sequences. Since these interspersed repeats could generate deletions and insertions by mispairing, rearrangements of this gene are a possibility in those FA families in which FAA mutations have not been identified.

Base Sequence↗
PubMed1992

Molecular and functional characterization of the murine glucocerebrosidase gene.

A genomic clone of glucocerebrosidase (D-glucosyl-N-acyl-sphingosine glucohydrolase; E.C. 3.2.1.45) purified from a genomic library derived from a Balb/c mouse was analyzed by restriction mapping and nucleotide sequencing of its promoter and protein coding regions. Promoter activity was functionally assessed by ligation of a 2 kb glucocerebrosidase fragment to the protein coding segment of a bacterial neomycin resistance gene. Smaller segments of the 5' flanking sequence were then analyzed for their ability to initiate transcription of the chloramphenicol acetyltransferase reporter gene. A 319 bp Eco RI-Bgl II fragment (containing 259 bp upstream of the cDNA 5' limit) ligated to the chloramphenicol acetyltransferase open reading frame produced considerable activity.

3T3 Cells↗
PubMed1992

Molecular cloning of the breakpoints of a complex Philadelphia chromosome translocation: identification of a repeated region on chromosome 17.

Complex translocations in chronic myelogenous leukemia involve various chromosomes, in addition to chromosomes 9 and 22, in a nonrandom fashion. We have analyzed the DNA from leukemia cells characterized by a complex translocation, t(9;22;10;17)(q34;q11;p13;q21), by using the techniques of Southern blot hybridization, in situ hybridization, and molecular cloning; one of the breakpoints is at 17q21, a band that is frequently involved in complex 9;22 translocations. All of the breakpoint junctions and the corresponding normal sequences from the four involved chromosomes have been molecularly cloned. Restriction mapping is consistent with a simple concerted exchange of chromosomal material among the four chromosomes, except that additional changes appeared to have occurred within the chromosome 17 sequences. The cloned sequences on chromosome 17 at band q21 were found to be repeated in normal cells. By fluorescence in situ hybridization, a strong signal is seen at 17q21, but a weaker signal is also present at 17q23. By comparison with other primate species, an inversion in chromosome 17 during evolution appears to be responsible for the splitting of the cluster of repeat units in normal human cells.

Animals↗
PubMed1994

Polydnavirus of the parasitic wasp Chelonus inanitus (Braconidae): characterization, genome organization and time point of replication.

Ultrastructural analysis of the polydnavirus of the braconid wasp Chelonus inanitus revealed that virions consist of one cylindrical nucleocapsid enveloped by a single unit membrane. Nucleocapsids have a constant diameter of 33.7 +/- 1.4 nm and a variable length of between 8 and 46 nm. Spreading of viral DNA showed that the genome consists of circular dsDNA molecules of variable sizes and measurement of the contour lengths indicated sizes of between 7 and 31 kbp. When virions were exposed to osmotic shock conditions to release the DNA, only one circular molecule was released per particle suggesting that the various DNA molecules are singly encapsidated in this bracovirus. The viral genome was seen to consist of at least 10 different segments and the aggregate genome size is in the order of 200 kbp. By partial digestion of viral DNA with HindIII or EcoRI in the presence of ethidium bromide and subsequent ligation with HindIII-cut pSP65 or EcoRI-cut pSP64 and transfection into Escherichia coli, libraries of 103 HindIII and 23 EcoRI clones were obtained. Southern blots revealed that complete and unrearranged segments were cloned with this approach, and restriction maps for five segments were obtained. Part of a 16.8 kbp segment was sequenced, found to be AT-rich (73%) and to contain six copies of a 17 bp repeated sequence. The development of the female reproductive tract in the course of pupal-adult development of the wasp was investigated and seen to be strictly correlated with the pigmentation pattern. By the use of a semiquantitative PCR, replication of viral DNA was observed to initiate at a specific stage of pupal-adult development.

Animals↗
PubMed1993

Isolation and characterization of cDNA clones for chicken major histocompatibility complex class II molecules.

The chicken major histocompatibility complex (MHC), the B complex, is being intensively analysed at the DNA level. To further probe the molecular structure of chicken MHC class II genes, cDNA clones coding for chicken MHC class II (B-L) beta chain molecules were isolated from an inbred G-B2 Leghorn chicken spleen and liver. Twenty-nine cDNA clones were isolated from the spleen and eight cDNA clones were isolated from the liver. Based on restriction maps, most clones could be clustered into one family of genes. Four cDNA clones were sequenced (S7, S10 and S19 from the spleen and L1, which was identical to S19, from the liver). Complete amino acid sequences of B-L beta chain molecules were predicted from the nucleotide sequences of the cDNA clones. Although both the nature and the location of the conserved residues were similar in chicken and mammalian sequences, some species-specific differences were found, suggesting that the structures of the B-L molecules of this haplotype are similar, but not identical, to their mammalian counterparts.

Amino Acid Sequence↗
PubMed1998

Isolation of genomic DNA containing a cytosolic ascorbate peroxidase gene (ApxSC) from the strawberry (Fragaria x ananassa).

We isolated a genomic DNA harboring a cytosolic ascorbate peroxidase gene (ApxSC) from a genomic library of the strawberry (Fragaria x ananassa). Restriction mapping and sequence analyses showed that the DNA is composed of 2.5 kb of the full-length ApxSC gene, 3.7 kb of the 5'-upstream region, and 0.5 kb of the 3'-downstream region. The ApxSC genomic DNA contains 10 exons and 9 introns, which is similar to the structure of pea ApxI. A primer extension analysis suggested that the transcription of ApxSC gene was started at three start sites with different degrees. The promoter region of ApxSC gene contains a sequence or structure distinct from other reported plant ascorbate peroxidase genes, though with several known functional elements such as a TATA box.

Amino Acid Sequence↗
PubMed1992

Sequence complementarity between the 5'-terminal regions of mRNAs for rat mitochondrial cytochrome P-450c27/25 and a growth hormone-inducible serine protease inhibitor. A possible gene overlap.

Recently we reported that a P-450c27/25 cDNA probe hybridizes to two RNA species of about 1.9 and 2.3-2.4 kilobase pairs (kb) in some rat tissues. To understand the molecular relationship between the two mRNAs, we have isolated and characterized a cDNA for the larger, previously uncharacterized 2.3-kb mRNA species. The 2.3-kb cDNA is identical to the previously reported 1.9-kb P-450c27/25 cDNA excepting a 400-nucleotide-long 5' extension. The terminal 291 nucleotides of this extension exhibit 100% complementarity with the 5'-translated region of the mRNA belonging to a family of growth hormone-inducible serine protease inhibitors (SPI). Northern blot analysis, using strand-specific probes, and S1 nuclease protection revealed the presence of the 2.3-kb mRNA exhibiting the sequence characteristics of the larger cDNA. These results were further confirmed by polymerase chain reaction amplification of reverse transcribed RNA. Expression of the 2.3-kb cDNA in COS cells resulted in the correct mitochondrial targeting of a 52-kDa protein exhibiting the properties of P-450c27/25. Furthermore, both the 1.9- and 2.3-kb mRNAs appear to direct the synthesis of a similarly sized 55-kDa precursor protein in a reticulocyte lysate system. Restriction mapping, polymerase chain reaction amplification and partial sequencing of a 25-kb genomic DNA clone suggest the proximal location of the SPI and the P-450c27/25 protein coding regions in the rat genome on either side of a common overlap region. The results also show that the P-450c27/25 mRNAs are regulated by growth hormone in parallel to the SPI mRNAs. These results collectively suggest that a growth hormone-inducible SPI family mRNA and the P-450c27/25 mRNA are encoded by two closely linked, possibly overlapping genes.

Amino Acid Sequence↗
PubMed1991

Characterization of new plasmids from methylotrophic bacteria.

Several tens of methanol-utilizing bacterial strains isolated from soil were screened for the presence of plasmids. From the obligate methylotroph Methylomonas sp. strain R103a plasmid pIH36 (36 kb) was isolated and its restriction map was constructed. In pink-pigmented facultative methylotrophs (PPFM), belonging to the genus Methylobacterium four plasmids were detected: plasmids pIB200 (200 kb) and pIB14 (14 kb) in the strain R15d and plasmids pWU14 (14 kb) and pWU7 (7.8 kb) in the strain M17. Because of the small size and the presence of several unique REN sites (HindIII, EcoRI, NcoI), plasmid pWU7 was chosen for the construction of a vector for cloning in methylotrophs. Cointegrates pKWU7A and pKWU7B were formed between pWU7 and the E. coli plasmid pK19 Kmr, which were checked for conjugative transfer from E. coli into the methylotrophic host.

Anti-Bacterial Agents↗
PubMed1993

Characterization of a cta/CDE operon-like genomic region encoding subunits I-III of the cytochrome c oxidase of the cyanobacterium Synechocystis PCC 6803.

Strong heterologous hybridization of a synthetic oligonucleotide probe of 17 bp originally used to clone subunit I of the Paracoccus denitrificans cytochrome c oxidase (M. Raitio, T. Jalli and M. Saraste (1987) EMBO J. 6, 2825-2833) to a single band was observed on Southern blots of Anacystis nidulans R2 (Synechococcus PCC 7942), Synechocystis PCC 6803, and Nostoc Mac PCC 8002 chromosomal DNA digests. Six pooled gene banks prepared from Synechocystis PCC 6803 contained regions that hybridized to the oligonucleotide (probe C) which is specifically directed toward the putative Cu-binding site VWAHHMY of subunit I. Two of these gene banks were transformed into Escherichia coli and screened for colonies hybridizing to probe C. Several clones were recovered, and one type of plasmid was identified from each gene bank. The two (overlapping) plasmids were called pDAUV1 and pDAUV2. A restriction map of the plasmids showed that the overlapping region contained an 80 bp PvuI-KpnI fragment binding to probe C. The two clones together permitted sequencing of the entire gene for cytochrome c oxidase subunit I from Synechocystis PCC 6803. Further systematic sequencing of approximately 1000 bp upstream and downstream each of the ctaD (subunit I) gene revealed the presence of two genes encoding subunits II (ctaC gene) and III (ctaE gene) due to conspicuous similarities to homologous genes from other cytochrome c oxidase-containing organisms. Yet, no indications of genes encoding additional subunits of the oxidase were found within the region sequenced.

Amino Acid Sequence↗