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At least 991 records · Page 55Linked to original sources
PubMed1989

Yeast genomic clones encoding polypeptides immunologically related to an 18 kDa subunit of mitochondrial ATP synthase.

A yeast genomic library in the bacteriophage expression vector lambda gt11 was screened with a polyclonal anti-holo-ATPase antiserum resulting in the isolation of 54 immunoreactive clones. Four of these phage clones express in bacteria a polypeptide antigenically related to an 18 kDa subunit (P18) of the yeast mitochondrial ATPase complex. Molecular analysis of the yeast DNA inserts in these phage clones revealed two classes of yeast DNA that share little homology at the nucleotide sequence level and therefore may represent distinct separate genes. The polypeptides potentially encoded by these yeast DNA segments do show scattered short blocks of strong amino acid sequence homology, which may underlie the observed immunochemical relatedness between the proteins expressed in bacteria.

ATP Synthetase Complexes↗
PubMed1994

[Molecular cloning of the adherence gene cluster from uropathogenic Escherichia coli and preparation of its antisera].

A genomic library of the uropathogenic E. coli J96 was constructed by using cosmid pHC79 as cloning vector. Two positive recombination cosmids which could express the adherence characters were acquired. From both cosmids a EcoRI fragment was subcloned into the vector pACYC184 by shot-gun method. Three colonies were found which exhibited MRHA and production of P Pili. One of them, pCT10/E. coli K-12 P678-54, was about 14.6 kb and was used to prepare the antisera. After absorption with pACYC184/E. coli K-12 P678-54 for three times, the antisera were revealed specific against the adherence gene cluster of uropathogenic E. coli by the SDS-PAGE and Western blotting of the P pilus crude extracts and the hemagglutination inhibition test.

Adhesins, Bacterial↗
PubMed2001

Selection of virulence-associated determinants of Streptococcus suis serotype 2 by in vivo complementation.

Within Streptococcus suis serotype 2, pathogenic, weakly pathogenic, and nonpathogenic strains can be found. We introduced a genomic library of a pathogenic strain into a weakly pathogenic strain. After infection of the library into young piglets pathogenic transformants were selected. One specific transformant containing a 3-kb fragment of the pathogenic strain appeared to be dominantly enriched in diseased pigs. The observed enrichment was not tissue specific. The selected fragment, when introduced into two different weakly pathogenic strains, increased the virulence of these strains considerably. In contrast, introduction of the corresponding fragment of a weakly pathogenic strain had only minor effects on virulence. Nucleotide sequence analysis of the selected fragment of the pathogenic strain revealed the presence of two potential open reading frames, both of which were found to be mutated in the corresponding fragment of the weakly pathogenic strain. These data strongly suggest that the selected fragment contains determinants important for virulence.

Amino Acid Sequence↗
PubMed1993

Genomic cloning, heterologous expression and pharmacological characterization of a human histamine H1 receptor.

A human histamine H1 receptor gene lacking introns was isolated by screening a human genomic library with a bovine histamine H1 receptor probe. The deduced protein of 487 amino acids showed characteristic properties of G-protein-coupled receptors. The coding region was subcloned into the expression vector pSVL (Pharmacia), and the resulting construct transfected into COS-7 cells. Binding studies with [3H]pyrilamine on membranes from transfected cells revealed saturable specific binding with a KD of 1.2 nM and a Bmax of 3400 fmol/mg protein. Binding affinities of histamine and known histamine antagonists were similar to those for histamine H1 receptors in guinea-pig cerebellum. In transfected COS-7 cells, histamine induced inositol phosphate formation, that was inhibitable by pyrilamine.

Amino Acid Sequence↗
PubMed1998

Isolation and characterization of the Xanthomonas campestris rpoH gene coding for a 32-kDa heat shock sigma factor.

Degenerate oligonucleotide primers corresponding to the conserved regions of bacterial heat shock sigma factor RpoH (sigma 32) were used to amplify a 190-bp fragment by PCR on the X. campestris pv. campestris strain 11 chromosome. Using this fragment as a probe, plasmid pXC57 carrying a 4.7-kb insert was isolated from a genomic library of Xc11. Sequence analysis of a stretch of 2,053 bp from the pXC57 insert revealed an ORF encoding a polypeptide of 291 aa (32,854 dal) which displays 59.6% and 57.3% identity to the rpoH gene products of E. coli and P. aeruginosa, respectively. The Xc11 rpoH gene was able to complement the RpoH deficient E. coli strain A7448. Both amino acid and mRNA sequences deduced from the Xc11 rpoH gene show structural features characteristics of the corresponding sequences from those of the gamma subgroup proteobacteria. The RpoH levels in Xc11 were demonstrated to increase transiently in response to heat shock treatment by immunoblot analysis using the polyclonal antibody raised against the purified Xc11 RpoH.

Amino Acid Sequence↗
PubMed1995

A Bkm-associated human y-chromosomal DNA is conserved and transcribed in the testis of mouse.

Y chromosome associated genes and repetitive sequences are continually viewed from the point of view of their possible involvement in sex determination and in the evolution of such a mechanism, thus sustaining an interest in the identification of novel sequences to gain newer insights. Here we have used the highly conserved class of Bkm repeats to isolate its associated sequences from the Y chromosome under the assumption that these sequences could be involved in sex determination and might also reflect the evolutionary status of the Y chromosome. Towards this end we have screened a genomic library enriched with human Y chromosome DNA with Bkm. One of the positive clones, C65, has a pericentromeric location on the Y chromosome and is present in a number of human sex-reversed XX males. The 10.5kb insert of clone C65 has been further subcloned (pFI, pFII, pFIII, pFIV). The subclone pFIII is present in both sexes in human and mouse, whereas pFIV is primate specific and present in both sexes. pFII contains sequences homologous to Bkm. pFI is conserved in mouse and man, but is Y specific only in primates. Although present in both sexes in mouse, pFI is transcribed specifically in the male testis suggesting that it may be involved in the process of sex determination or testis differentiation and spermatogenesis.

Animals↗
PubMed1991

Actin in the oomycetous fungus Phytophthora infestans is the product of several genes.

Actin (ACT) in Phytophthora infestans is encoded by at least two genes, in contrast to unicellular and other filamentous fungi where there is a single gene. These genes (designated actA and actB) have been isolated from a genomic library of P. infestans. The complete nucleotide sequence of both genes has been determined. Unlike the actin-encoding genes (act) of other filamentous fungi, no introns are obvious in the coding region, a feature shared with the act genes of certain protists. Northern blotting and primer extension studies of the mRNA show that actA and actB are actively transcribed in mycelium, sporangia and germinating cysts but only at a low level in the case of actB. Both genes display bias in their codon usage. This is more extreme in actA. The deduced ACTB protein is strikingly similar to that of the Phytophthora megasperma actin and is more diverged from other actins than ACTA.

Actins↗
PubMed1997

DNA fingerprinting of Campylobacter fetus using cloned constructs of ribosomal RNA and surface array protein genes.

DNA fragments coding for the ribosomal RNA and the surface array proteins of Campylobacter fetus have been cloned from a genomic library constructed in Escherichia coli. They were used in the molecular characterization of C. fetus (subsp. fetus; subsp. venerealis) strains by restriction fragment length polymorphism (RFLP) method. Ribotyping results showed that all strains of the two subspecies can be classified under one ribogroup implying very close relatedness. The sapA gene DNA marker, however, discriminated all the strains regardless of the subspecies when chromosomal DNA was restricted with HindIII, HaeIII, XbaI or EcoRV. These results illustrate that the sapA probe is potentially useful in fingerprinting C. fetus strains and in determining the relationships of strains for epidemiological purposes.

Animals↗
PubMed2000

Identification of a substrate for Pkn2, a protein Ser/Thr kinase from Myxococcus xanthus by a novel method for substrate identification.

Eukaryotic cells contain a large number of protein Ser/ Thr kinases, which play important roles in signal transduction required for cell proliferation, differentiation, and stress response and adaptation. It is also known that some prokaryotes contain a family of protein Ser/Thr kinases. A major challenge in the characterization of these kinases is how to identify their specific substrates. Here we developed such a method using a protein Ser/Thr kinase, Pkn2 from Myxococcus xanthus, a Gram-negative soil bacterium. When Pkn2 is inducibly expressed in E. coli, cells are unable to form colonies on agar plates. This lethal effect of Pkn2 was eliminated in an inactive Pkn2 mutant in which the highly conserved Lys residue was changed to Asn, indicating that phosphorylation of a cellular protein(s) in E. coli resulted in growth arrest. Several clones from an E. coli genomic library were found to suppress the lethal effect when co-expressed with pkn2. Four out of seven multi-copy suppressors were identified to encode HU, (3 for HUalpha and 1 for HUB) a histone-like DNA binding protein. Purified HUalpha was found to be specifically phosphorylated by Pkn2 at Thr-59, and the phosphorylated HUalpha became unable to bind to DNA, suggesting that the phosphorylation of endogenous HU proteins by Pkn2 contributed at least in part to the lethal effect in E. coli. The present method termed the STEK method (Suppressors of Toxic Effects of Kinases) may be widely used for the substrate identification not only for prokaryotic protein Ser/Thr kinases but also for eukaryotic kinases.

Bacterial Proteins↗
PubMed1996

Structural organization of the rae28 gene, a putative murine homologue of the Drosophila polyhomeotic gene.

A putative murine homologue of the Drosophila polyhomeotic gene, named rae28, has been isolated from a genomic library of 129/SV mouse and its structural organization has been analyzed. rae28 is a single gene of approximately 22 kb long and consists of 15 exons. Its 5'-flanking region lacks typical transcriptional regulatory sequences, such as TATA and CCAAT boxes, but contains GC-rich sequences and seven putative binding sites for a transcription factor, Sp1. One major transcription start point has been determined. The overall exon-intron organization suggested that three different Rae28 mRNAs are generated through alternative splicing. Furthermore, the rae28 gene has been located on the R-positive F3 band of mouse chromosome 6 by the direct R-banding fluorescence in situ hybridization methods.

Animals↗
PubMed2001

Cloning and characterization of a bacterial cell-bound type B carboxylesterase from Bacillus sp. BP-7.

A clone producing halos on tributyrin plates was isolated from a genomic library of Bacillus sp. BP-7. The insert contained an open reading frame that coded for a protein of 487 amino acids with homology to carboxylesterases. The cloned enzyme showed clear preference for esters of short-chain fatty acids, being classified as an esterase. Maximum activity was found at 45 degrees C and pH 7.5. The enzyme displayed stability in the pH range from 6 to 9.5, and at temperatures from 4 degrees to 45 degrees C. Zymogram analysis of the protein revealed a molecular mass of 53 kDa and a pI of 5.1. The enzyme showed homology to members of the bacterial subclass of type B carboxylesterases, a set of proteins potentially useful for biotechnological applications.

Amino Acid Sequence↗
PubMed1997

Cloning, sequence, and expression of the L-(+) lactate dehydrogenase of Streptococcus bovis.

The ldh gene encoding the fructose-1,6-diphosphate-dependent L-(+) lactate dehydrogenase from the ruminal bacterium Streptococcus bovis was cloned and sequenced. A genomic library of S. bovis JB1 DNA was constructed in lambda ZAP II and screened by use of a heterologous probe derived from the cloned Streptococcus mutans ldh gene. Several clones were isolated that contained a common 2.9-kb fragment as determined by restriction analysis. Nucleotide sequence analysis revealed a 987-bp open reading frame with extensive homology to Streptococcus thermophilus and S. mutans ldh nucleic acid and amino acid sequences. Expression of the cloned S. bovis ldh gene in Escherichia coli was confirmed by the ability to complement the ldh mutation of E. coli FMJ39, by using an in-gel activity screen and by enzymatic assay. Increased LDH activity was observed in S. bovis JB1 containing the cloned ldh genes on a multicopy plasmid.

Amino Acid Sequence↗
PubMed2004

Identification of a novel gene hbrB required for polarised growth in Aspergillus nidulans.

We have cloned a novel gene, hbrB, by complementation of a temperature sensitive hyperbranching (hbr) mutant of Aspergillus nidulans. The mutant, hbrB3, exhibits hyperseptation and shows a marked increase in hyphal branching at the restrictive temperature. A genomic library incorporating the AMA1 sequence, which confers autonomous replication on the plasmid, was used to clone the gene. Co-ordinate loss of the complementing plasmid and wild type phenotype was shown. The 847 amino acid predicted product of the hbrB gene shows significant identities with Neurospora crassa and Aspergillus fumigatus hypothetical proteins but no other obvious relative outside the filamentous fungi. The gene was placed under control of the alcA promoter and downregulation resulted in a loss of polarity phenotype. These results suggest that A. nidulans hbrB is specific to filamentous fungi and is essential for hyphal growth and polarity. Index descriptors: Aspergillus nidulans; branching; hbrB; hyphal growth and polarity

Artificial Gene Fusion↗
PubMed1992

Structure and expression of the rat epididymal secretory protein I gene. An androgen-regulated member of the lipocalin superfamily with a rare splice donor site.

The complete rat epididymal secretory protein I (ESP I) gene was isolated from a genomic library constructed in bacteriophage lambda Charon 4A. The complete nucleotide sequence of the gene and its immediate 5' and 3' flanking sequences were determined. Interesting features include the presence of a rare, but functional, splice donor site (...GC) and the presence of a putative androgen-receptor-binding element. A detailed analysis of ESP I regulation was carried out after castration and subsequent testosterone treatment, demonstrating the requirement for androgens. Efferent-duct ligation and cryptorchism, on the other hand, had no effect on the steady-state concentrations of ESP I transcripts. Comparison of the exon/intron organization of the ESP I gene with those of members of the lipocalin superfamily provides strong support for a common ancestral origin.

Amino Acid Sequence↗
PubMed1998

Cloning and sequencing of the homologues of both the bacterial and eukaryotic initiation factor genes (hIF-2 and heIF-2 gamma) from archaeal Halobacterium halobium.

The cloning and sequencing of the genes encoding the translational initiation factors (hIF-2 and heIF-2 gamma) was performed by screening the halophilic archaeon Halobacterium halobium genomic library with a probe constructed from the peptide IGHVDHGK that is conserved in archaeal GTP-binding elongation factors. The codon usage by the hIF-2 and heIF-2 gamma genes showed a preference for triplets ending in G or C. This characteristic is almost identical to that of other H. halobium genes. The translated protein of hIF-2 and heIF-2 gamma genes is made of 414 and 583 amino acid residues, respectively, and contains the sequence motif for the binding of GTP. The sequence of hIF-2 shows a strong similarity to the initiation factor IF-2 from Bacteria whereas heIF-2 gamma shows a strong similarity to the initiation factor eIF-2 gamma from Eucarya.

Amino Acid Sequence↗
PubMed1994

A family of U1 pseudogenes in Bombyx mori may be derived from an ancestral pseudogene.

Seven EMBL-4 lambda clones containing U1 small nuclear RNA sequences were isolated from a Bombyx mori genomic library. Six of the seven represent unique sequences. The six unique U1 sequences exhibit fixed point 3'-end truncation. Five out of the six clones share immediate 3'-end flanking sequences while two share 5'-end flanking sequences. Fixed point 3'-end truncation and a hierarchy of shared to unique diagnostic mutations may suggest a family of U1 pseudogenes generated from a reverse-transcribed class II pseudogene in B. mori. An ancestral 'master' U1 pseudogene capable of RNA- and/or DNA-mediated transposition may give rise to generations of U1 pseudogenes that include the original pseudogene's flanking sequences. Identical 3'-end truncation in some of these U1 sequences can be explained by RNA self-priming due to intra-strand binding prior to reverse transcription.

Animals↗
PubMed1992

Rearrangement of the human tre oncogene by homologous recombination between Alu repeats of nucleotide sequences from two different chromosomes.

The rearranged region of the tre oncogene originating from chromosomes 5q23q31 and 18q12 was cloned from tumor genomic DNA, sequenced and aligned with wild-type sequences cloned from a normal human genomic library. In the breakpoint region each wild-type sequence contained two Alu repeats. The recombination occurred between the 3'-most Alu from chromosome 5 and the 5'-most Alu from chromosome 18 and, consequently, resulted in a hybrid Alu flanked with one Alu on either side. The recombinant joint was located to a 20-bp homology region in left arms of the Alu repeats involved in recombination. The same homology region was identified in the hybrid Alu of the rearranged tre. At its 5' extremity the homology region overlaps the B box of Alu-borne RNA polymerase III promoter. The 100% identity score in the region of homology suggests that the recombination process was conservative and not error prone.

Base Sequence↗
PubMed1991

The phosphoenolpyruvate carboxylase gene family of Sorghum: promoter structures, amino acid sequences and expression of genes.

Two different members of the phosphoenolpyruvate carboxylase(PEPC)-encoding multigene family (clones lambda CP21 and lambda CP46) have been isolated from a Sorghum vulgare lambda EMBL4 genomic library. The use of the 3'-noncoding regions to probe Northern blots of RNA from roots, etiolated leaves and green leaves indicated that lambda CP21 and lambda CP46 encode the C3- and C4-type leaf PEPC isoforms, respectively. The lambda CP21 clone is expressed in the three tissues and is not light-regulated, whereas lambda CP46 is only expressed in greening leaves. The nucleotide sequence of the 5'-flanking DNA (520 bp) has been determined for both genes. For lambda CP46, several direct repeats were located in this region with similarities to sequences found in other light-regulated genes, but not in lambda CP21. The deduced amino acid sequences of the two S. vulgare PEPC proteins are 75% identical.

Amino Acid Sequence↗