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At least 1,189 records · Page 66Linked to original sources
PubMed1991

Cloning of three endoglucanase genes from Thermomonospora curvata into Escherichia coli.

A BamHI genomic library from Thermomonospora curvata was constructed in E. coli using cosmid vector pHC79. Four clones able to hydrolyze CMC were isolated. Restriction digests and Southern gel analysis revealed the presence of three different endoglucanase genes. DNA fragments contained in all of the endoglucanase cosmids hybridized to T. curvata chromosomal DNA. The cellulase genes were expressed in E. coli, but at rather low levels.

Bacteria↗
PubMed1991

Integrated maps of the Drosophila genome: progress and prospects.

A physical map of the Drosophila melanogaster genome is being assembled, consisting of ordered overlapping cosmid clones. The map is constructed in steps, separately for each chromosomal division. Gaps in this map are to be bridged with yeast artificial chromosome clones. Hybridization to previously cloned genes and extensive use of in situ hybridization to polytene chromosomes ensure that the cosmid map is firmly anchored to the wealth of available genetic and cytogenetic information. The intention is to make the physical map widely available as part of an overall, integrated genetic resource for the Drosophila research community.

Animals↗
PubMed1991

Orientation of HLA-DNA gene and identification of a CpG island-associated gene adjacent to DNA in human major histocompatibility complex class II region.

We have constructed the detailed physical map of the HLA class II gene region by the pulsed field gel electrophoresis (PFGE) and cosmid walking technique. In this process, the DNA gene was found to be located telomeric to DPA1 with the 5'----3' orientation which is the same as the DPA1 and DPA2 genes, but opposite to the DQA1, DQA2 and DRA genes. This orientation is reverse to that of the counterpart gene in the rabbit major histocompatibility complex region. About 30 kb downstream from the DNA gene towards DOB, a CpG island characterized by clustered sites for rare cutting restriction enzymes and frequently associated with the 5' end of housekeeping genes was identified by PFGE and cosmid walking. From a complementary DNA (cDNA) library constructed from a Epstein-Barr virus-transformed B-cell line, a cDNA clone was isolated using the genetic probe from this CpG island. Its nucleotide sequences suggested that it represented a new non-HLA gene with a single copy which was of little genetic polymorphism and named NAT (DNA-associated transcript). Northern blot analysis showed that the NAT gene was expressed with a 4-kb transcript in all of tissues examined so far.

Base Sequence↗
PubMed1989

Cloning of chromosomal genes of Lactococcus by heterologous complementation: partial characterisation of a putative lactose transport gene.

A cosmid gene library of the genome of Lactococcus lactis subsp. lactis 712 has been constructed in the broad host range plasmid pLAFR1 in Escherichia coli LE392. Three lactococcal genes from the bank were identified by heterologous complementation of specific mutations in strains of E. coli. A cosmid clone encoding a putative lactose transport gene was identified by complementing an E. coli lacY mutant. The complemented clone supported the uptake of 14C lactose in transport assays. The DNA fragment responsible was subcloned and localised to a 1.28 kb fragment of the lactococcal chromosome.

Biological Transport↗
PubMed1989

Cloning of the virulence regulatory (vir) locus of Bordetella pertussis and its expression in B. bronchiseptica.

Hybridisation with cosmid pRMB2, containing the vir locus of bordetella pertussis, showed that the Tn5 insertion in B. pertussis BP347 (Vir-) was located with 1 2.7 kB EcoRI fragment. When subcloned, this fragment alone was unable to complement BP347, but a larger 8.0 kb region which included the 2.7 kb EcoRI fragment, did restore expression of virulence associated properties to BP347, and to avirulent phase variants of both B. pertussis and B. bronchiseptica. EcoRI-digested DNA from stains of all four species of Bordetella showed homology to the cosmid genomic insert and to the 2.7 kb subclone, but B. avium showed a markedly different pattern of homology to the other species.

Blotting, Southern↗
PubMed1991

Suppression of an Escherichia coli secA(ts) mutant by a gene cloned from Staphylococcus carnosus.

Escherichia coli mutant MM52 (secA(ts)) was transformed with a cosmid library from Staphylococcus carnosus, and a recombinant cosmid (pBO23) allowing growth at the non-permissive temperature (42 degrees C) was isolated. pBO23 also restored the growth defects of E. coli mutants IQ85 (secY(ts)) and IT41 (lep(ts)). Nucleotide sequencing revealed that the DNA fragment responsible for the suppression effect codes for a S. carnosus protein highly homologous to the ribosomal protein L13 of E. coli. The staphylococcal L13 protein was efficiently incorporated into E. coli ribosomes. Possible explanations for the effect of this polypeptide on the growth of temperature-sensitive E. coli secretion mutants are discussed.

Adenosine Triphosphatases↗
PubMed1991

Cloning and partial characterization of genes for ribosomal ribonucleic acid in Lactococcus lactis subsp. lactis.

A cosmid gene library of the genome of Lactococcus lactis subsp. lactis 712 was probed for the presence of 16S rRNA genes, using 32P 5' end-labelled 16S rRNA fragments. Cosmid DNA from positive clones responsible for hybridisation was subcloned into a high copy number vector and a restriction map was constructed. The location of the 16S, 23S and 5S rRNA genes was determined on this map. Transcriptional promoter activity was identified upstream of the 5' end of the 16S rRNA gene. By probing L. lactis 712 chromosomal DNA cut with a range of restriction endonucleases, with a conserved oligonucleotide to the 5' end of the 16S rRNA gene, 6 copies of rRNA genes were identified.

Base Sequence↗
PubMed1992

Cloning of poly(3-hydroxybutyric acid) synthase genes of Rhodobacter sphaeroides and Rhodospirillum rubrum and heterologous expression in Alcaligenes eutrophus.

From genomic libraries of the purple non-sulfur bacteria Rhodospirillum rubrum Ha and Rhodobacter sphaeroides ATCC 17023 in the broad-host range cosmid pVK100, we cloned a 15- and a 14-kbp HindIII restriction fragment, respectively. Each of these fragments restored the ability to accumulate poly(3-hydroxybutyrate) (PHB), in the PHB-negative mutant Alcaligenes eutrophus PHB-4. These hybrid cosmids also complemented PHB-negative mutants derived from wild-type R. rubrum or R. sphaeroides. Both fragments hybridized with the PHB synthase structural gene of A. eutrophus H16 and conferred the ability to express PHB synthase activity. Only the 15-kbp HindIII fragment from R. rubrum conferred on the mutant PHB-4 the ability to form large PHB granules (length up to 3.5 microns).

Acyltransferases↗
PubMed1987

Isolation and characterization of the porcine growth hormone gene.

A cosmid clone containing the entire porcine growth hormone (PGH) gene has been isolated using a full-length PGH cDNA as the hybridization probe. The gene within the cosmid was subcloned into plasmids and completely sequenced. The coding, promoter, and both 5'- and 3'-noncoding sequences of the PGH gene were found to be highly conserved when compared to the previously sequenced genes coding for rat, human and bovine growth hormones, and also to the human placental lactogen gene. The high degree of conservation between the 5'- and 3'-noncoding regions of the genes from these different species indicates that growth hormone genes may be evolving by some unusual mechanism. The PGH gene was found to contain the unusual variant GC donor splice site.

Amino Acid Sequence↗
PubMed1988

Tandem gene amplification in vitro for rapid and efficient expression in animal cells.

Plasmid vectors pHSG293 and pHSG747, suitable for in vitro gene amplification for subsequent animal-cell expression, were developed. A cosmid vector pHSG293 confers Km resistance to Escherichia coli host cells and G418 resistance to animal cells and contains a single BstXI recognition/cleavage site, CCACGGGG/CTGG, near the cos site (the recognition site is underlined). The cassette vector plasmid pHSG747 contains a multiple cloning site (MCS) between the simian virus 40 early promoter and the poly(A) signal sequence flanked by the same BstXI sites and confers Cm resistance to E. coli host cells. After inserting a coding fragment for human protein C or its derivative in the appropriate orientation in the MCS of pHSG747, the BstXI expression unit fragment was purified, mixed with BstXI-digested pHSG293 DNA at a molecular ratio of 20 to 40:1 and ligated. This allowed for tandem gene amplification due to asymmetric cohesive ends. Ligation products were packaged in lambda phage particles, amplified in E. coli cells as large cosmid molecules, and then introduced into CHO cells. G418R transformants were found to produce and secrete recombinant protein molecules at a high level. The plasmid vectors developed in this work will provide a rapid screening system useful for protein engineering in animal cells.

Animals↗
PubMed1988

Ammonia-constitutive nitrogen fixation mutants of Rhodobacter capsulatus.

Mutants of R. capsulatus that express nif genes constitutively with respect to ammonia were studied in order to define better the circuit that regulates nif gene transcription. One mutant class could be complemented in trans by a cosmid clone containing a wild-type gene (nifR5) defined by Tn5 inserts as being no longer than 1.6 kb. The nifR5 gene is unlinked to previously described nif genes. A second mutant class could not be complemented by the wild-type cosmid library. For one mutant in this class, a nifH::lac fusion was used to select further mutants that were Lac-. Only two of these could be complemented in trans to Lac+; the complementing gene was nifR4, which is analogous to the ntrA gene of enterobacteria. Both complemented strains were Nifc. Therefore these mutations do not bypass the need for the nifR4 gene product. A third class of constitutive mutant was found by selecting Nif+ revertants of a Nif- strain deleted for the nifR1 and nifR2 genes. The nifR1 and nifR2 genes are homologues of enterobacterial ntrC and ntrB genes, respectively. Not all of the Nif+ revertants were constitutive; some were regulated normally by ammonia. We suspect that the latter revertants use alternate Ntr systems to activate nif gene transcription, a suggestion consistent with the observation that numerous bands in Southern blots of total DNA of R. capsulatus are identified by Escherichia coli ntr gene probes.

Ammonia↗
PubMed1988

The transcriptionally active human ribosomal protein S17 gene.

A human ribosomal protein S17 cDNA [Chen et al., Proc. Natl. Acad. Sci. USA 83 (1986) 6907-6911] was used to isolate four S17 DNA clones from human genomic libraries constructed in bacteriophage lambda and cosmid vectors. Based on its transcriptional activity in a transient expression assay and on sequence similarity with S17 cDNA, cosmid clone HGS17-6 was identified as carrying the functional RPS17 gene. RPS17 is composed of five exons and four introns that span 4 kb of DNA. Two lambda clones of human genomic DNA were recognized as containing processed S17 pseudogenes, because (i) they were transcriptionally inactive in the transient assay, and (ii) they possess multiple, perfectly spliced RPS17 exons. Their coding sequences differ slightly from the cDNA and functional genomic clone.

Animals↗
PubMed1988

Cloning and determination of the nucleotide sequence of the Mn-containing superoxide dismutase gene from Halobacterium halobium.

A group of synthetic 17-mer oligodeoxynucleotides (oligos) was constructed to correspond to a sequence of amino acids situated near the N terminus of the manganese-containing superoxide dismutase (Mn-SOD) purified from the halophilic bacterium, Halobacterium halobium. A cosmid library of a Sau3AI partial digest of halobium DNA, cloned into the BamHI site of pHC79, was probed with the radiolabeled oligos. Cosmid DNA was purified from the clone that showed hybridization at the highest stringency. A 1.8-kb PstI fragment of this DNA which hybridized the probes was subcloned into bacteriophage M13 and transfected into Escherichia coli JM101. The entire insert containing a 600-bp sequence coding for Mn-SOD and its 5'- and 3'-flanking regions was sequenced. The derived amino acid sequence of the structural gene showed a similarity to other manganese and iron-containing SODs in normally conserved regions.

Amino Acid Sequence↗
PubMed1988

Improved broad-host-range plasmids for DNA cloning in gram-negative bacteria.

Improved broad-host-range plasmid vectors were constructed based on existing plasmids RSF1010 and RK404. The new plasmids pDSK509, pDSK519, and pRK415, have several additional cloning sites and improved antibiotic-resistance genes which facilitate subcloning and mobilization into various Gram-negative bacteria. Several new polylinker sites were added to the Escherichia coli plasmids pUC118 and pUC119, resulting in the new plasmids, pUC128 and pUC129. These plasmids facilitate the transfer of cloned DNA fragments to the broad-host-range vectors. Finally, the broad-host-range cosmid cloning vector pLAFR3 was improved by the addition of a double cos casette to generate the new plasmid, pLAFR5. This latter cosmid simplifies vector preparation and has permitted the rapid cloning of genomic DNA fragments generated with Sau3A. The resulting clones may be introduced into other Gram-negative bacteria by conjugation.

Amino Acid Sequence↗
PubMed1989

Nucleotide sequence of the structural gene, tcpA, for a major pilin subunit of Vibrio cholerae.

The toxin co-regulated pilus (Tcp) of Vibrio cholerae appears to be a major protective antigen. By cosmid cloning we have isolated a number of clones capable of converting Tcp- El Tor strains of V. cholerae to Tcp+. A synthetic oligodeoxyribonucleotide probe based upon the N-terminal amino acid sequence of TcpA, has been used to localize the structural gene within the cosmid clones. Using suitable subclones, the nucleotide sequence of the tcpA gene has been determined. The gene encodes a 23.3-kDa pre-protein which in its mature form has a size of 20.3 kDa. The N-terminal leader peptide or signal sequence is atypical and does not conform with the usual rules of such sequences. The TcpA protein shows some similarities to the major pilins of the methylated phenylalanine type or type-4 pili from other bacteria; however, it is sufficiently different that it may represent a new class.

Amino Acid Sequence↗
PubMed1991

A series of wide-host-range low-copy-number vectors that allow direct screening for recombinants.

A series of controlled expression vectors was constructed based on the wide-host-range plasmid pMMB66EH. Some of these new vectors code for the alpha-peptide of beta-galactosidase and allow the direct screening of recombinant clones by inactivation of alpha-complementation. The bla gene was replaced in some plasmids by the cat gene of Tn9 coding for chloramphenicol resistance, extending the use into beta-lactam-resistant strains. They all feature either the tac or taclac (tac-lac UV5 in tandem) promoters in front of a polylinker followed by the rrnB transcriptional stop point. These vectors were tested by subcloning the xylE gene coding for the Pseudomonas putida catechol 2,3-oxygenase and the Escherichia coli lamB gene coding for the lambda receptor. The expression of these genes in E. coli indicated that the tac promoter is five times stronger than the taclac promoter and that both were tightly regulated. The tac promoter in Pseudomonas syringae pv glycinea and Xanthomonas campestris pv vesicatoria had a strength similar to that in E. coli, while the taclac promoter was much weaker, reaching only 6.5 and 3% of the level of expression of the tac promoter, respectively. The taclac promoter, however, proved to be useful for the cloning in E. coli of DNA fragments that were unstable in vectors with stronger promoters and higher copy number. Expression of the lamB gene in Vibrio cholerae strain TRH7000 was not sufficient to permit cosmid transduction. Two subunits of the E. coli mannose permease, coded by the ptsP and ptsM genes, are also required for cosmid DNA penetration into the recipient cells.

Amino Acid Sequence↗
PubMed1993

Escherichia coli host strains SURE and SRB fail to preserve a palindrome cloned in lambda phage: improved alternate host strains.

We have attempted to produce Escherichia coli strains with the optimal combination of host mutations required for the construction of genomic libraries in lambda and cosmid vectors. For lambda vectors, we defined this as a strain that combined high efficiency of phage plating with optimal tolerance to DNA methylation and the ability to propagate recombinants containing regions of potential secondary structure. To optimize this latter property, we have tested a series of strains for the ability to propagate a lambda phage containing a palindromic sequence. These included an mcr- derivative of a strain shown by Ishiura et al. [J. Bacteriol. 171 (1989) 1068-1074] to allow optimal stability of inserts in cosmid clones. All the sbcC strains allowed plaque formation of the palindrome-containing lambda phage. However, while the palindrome-containing phage plated with reasonable efficiency on SURE (recB sbcC recJ umuC uvrC) and SRB (sbcC recJ umuC uvrC), the majority of phage recovered from these strains no longer required an sbcC host for subsequent plating. These two strains also gave poorer titres with a low-yielding phage clone from the human Prader-Willi chromosome region. Optimal phage hosts appear to be those that are mcrA delta(mcrBC-hsd-mrr) combined with mutations in sbcC plus recBC or recD and without mutations in additional recombination functions such as recJ or recJ umuC uvrC (all of our E. coli strains are available on request).

Bacteriophage lambda↗
PubMed1995

Amplification and cloning of the Mycobacterium tuberculosis dnaA gene.

To identify and subsequently clone the gene encoding the DnaA protein, degenerate oligodeoxyribonucleotide (oligo) primers targeted against two highly conserved domains of the eubacterial DnaA were used to amplify a 780-bp DNA region spanning the two primers from genomic DNA preparations of Mycobacterium tuberculosis (Mt), M. bovis (Mb) and M. avium (Ma). Nucleotide (nt) sequences and deduced amino acid (aa) sequences of these fragments revealed homologies with each other and with the corresponding regions from other bacteria. Using an oligo specific to Mt dnaA as a probe, the Mt genomic DNA cosmid libraries propagated in Escherichia coli were screened and a cosmid DNA clone hybridizing with the oligo was identified. Furthermore, a 5-kb DNA fragment containing the Mt dnaA was subcloned into a pUC18 vector.

Amino Acid Sequence↗