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At least 1,369 records · Page 76Linked to original sources
PubMed1998

Expression cloning of a Pseudomonas gene encoding a hydroxydecanoyl-acyl carrier protein-dependent UDP-GlcNAc acyltransferase.

UDP-N-acetylglucosamine-3-O-acyltransferase (UDP-GlcNAc acyltransferase) catalyzes the first step of lipid A biosynthesis (M. S. Anderson and C. R. H. Raetz, J. Biol. Chem. 262:5159-5169, 1987). We here report the isolation of the lpxA gene of Pseudomonas aeruginosa from a library of Pseudomonas strain PAO1 expressed in Escherichia coli LE392 (J. Lightfoot and J. S. Lam, J. Bacteriol. 173:5624-5630, 1991). Pseudomonas lpxA encodes a 10-carbon-specific UDP-GlcNAc acyltransferase, whereas the E. coli transferase is selective for a 14-carbon acyl chain. Recombinant cosmid 1137 enabled production of a 3-hydroxydecanoyl-specific UDP-GlcNAc acyltransferase in E. coli. It was identified by assaying lysozyme-EDTA lysates of individual members of the library with 3-hydroxydecanoyl-acyl carrier protein (ACP) as the substrate. Cosmid 1137 contained a 20-kb insert of P. aeruginosa DNA. The lpxA gene region was localized to a 1.3-kb SalI-PstI fragment. Sequencing revealed that it contains one complete open reading frame (777 bp) encoding a new lpxA homolog. The predicted Pseudomonas LpxA is 258 amino acids long and contains 21 complete hexapeptide repeating units, spaced in approximately the same manner as the 24 repeats of E. coli LpxA. The P. aeruginosa UDP-GlcNAc acyltransferase is 54% identical and 67% similar to the E. coli enzyme. A plasmid (pGD3) containing the 1.3-kb SalI-PstI fragment complemented E. coli RO138, a temperature-sensitive mutant harboring lpxA2. LpxA assays of extracts of this construct indicated that it is > 1,000-fold more selective for 3-hydroxydecanoyl-ACP than for 3-hydroxymyristoyl-ACP. Mass spectrometry of lipid A isolated from this strain by hydrolysis at pH 4.5 revealed [M-H]- 1,684.5 (versus 1,796.5 for wild-type lipid A), consistent with 3-hydroxydecanoate rather than 3-hydroxymyristate at positions 3 and 3'.

Acyltransferases↗
PubMed1999

Cloning, sequencing, and characterization of the cgmB gene of Sinorhizobium meliloti involved in cyclic beta-glucan biosynthesis.

Periplasmic cyclic beta-glucans of Rhizobium species provide important functions during plant infection and hypo-osmotic adaptation. In Sinorhizobium meliloti (also known as Rhizobium meliloti), these molecules are highly modified with phosphoglycerol and succinyl substituents. We have previously identified an S. meliloti Tn5 insertion mutant, S9, which is specifically impaired in its ability to transfer phosphoglycerol substituents to the cyclic beta-glucan backbone (M. W. Breedveld, J. A. Hadley, and K. J. Miller, J. Bacteriol. 177:6346-6351, 1995). In the present study, we have cloned, sequenced, and characterized this mutation at the molecular level. By using the Tn5 flanking sequences (amplified by inverse PCR) as a probe, an S. meliloti genomic library was screened, and two overlapping cosmid clones which functionally complement S9 were isolated. A 3.1-kb HindIII-EcoRI fragment found in both cosmids was shown to fully complement mutant S9. Furthermore, when a plasmid containing this 3.1-kb fragment was used to transform Rhizobium leguminosarum bv. trifolii TA-1JH, a strain which normally synthesizes only neutral cyclic beta-glucans, anionic glucans containing phosphoglycerol substituents were produced, consistent with the functional expression of an S. meliloti phosphoglycerol transferase gene. Sequence analysis revealed the presence of two major, overlapping open reading frames within the 3.1-kb fragment. Primer extension analysis revealed that one of these open reading frames, ORF1, was transcribed and its transcription was osmotically regulated. This novel locus of S. meliloti is designated the cgm (cyclic glucan modification) locus, and the product encoded by ORF1 is referred to as CgmB.

Base Sequence↗
PubMed2000

Mapping of the Cryptococcus neoformans MATalpha locus: presence of mating type-specific mitogen-activated protein kinase cascade homologs.

In this study we investigated the relationship between the MATalpha locus of Cryptococcus neoformans and several MATalpha-specific mitogen-activated protein (MAP) kinase signal transduction cascade genes, including STE12alpha, STE11alpha, and STE20alpha. To resolve the location of the genes, we screened a cosmid library of the MATalpha strain B-4500 (JEC21), which was chosen for the C. neoformans genome project. We isolated several overlapping cosmids spanning a region of about 71 kb covering the entire MATalpha locus. It was found that STE12alpha, STE11alpha, and STE20alpha are imbedded within the locus rather than closely linked to the locus. Furthermore, three copies of MFalpha, the mating type alpha-pheromone gene, a MATalpha-specific myosin gene, and a pheromone receptor (CPRalpha) were identified within the locus. We created a physical map, based on the restriction enzyme BamHI, and identified both borders of the MATalpha locus. The MATalpha locus of C. neoformans is approximately 50 kb in size and is one of the largest mating type loci reported among fungi with a one-locus, two-allele mating system.

Chemoreceptor Cells↗
PubMed2001

Genetic characterization of the Klebsiella pneumoniae waa gene cluster, involved in core lipopolysaccharide biosynthesis.

A recombinant cosmid containing genes involved in Klebsiella pneumoniae C3 core lipopolysaccharide biosynthesis was identified by its ability to confer bacteriocin 28b resistance to Escherichia coli K-12. The recombinant cosmid contains 12 genes, the whole waa gene cluster, flanked by kbl and coaD genes, as was found in E. coli K-12. PCR amplification analysis showed that this cluster is conserved in representative K. pneumoniae strains. Partial nucleotide sequence determination showed that the same genes and gene order are found in K. pneumoniae subsp. ozaenae, for which the core chemical structure is known. Complementation analysis of known waa mutants from E. coli K-12 and/or Salmonella enterica led to the identification of genes involved in biosynthesis of the inner core backbone that are shared by these three members of the Enterobacteriaceae. K. pneumoniae orf10 mutants showed a two-log-fold reduction in a mice virulence assay and a strong decrease in capsule amount. Analysis of a constructed K. pneumoniae waaE deletion mutant suggests that the WaaE protein is involved in the transfer of the branch beta-D-Glc to the O-4 position of L-glycero-D-manno-heptose I, a feature shared by K. pneumoniae, Proteus mirabilis, and Yersinia enterocolitica.

Animals↗
PubMed1999

Characterization of Varicella-Zoster virus glycoprotein K (open reading frame 5) and its role in virus growth.

Varicella-zoster virus (VZV) is an alphaherpesvirus that is the causative agent of chickenpox and herpes zoster. VZV open reading frame 5 (ORF5) encodes glycoprotein K (gK), which is conserved among alphaherpesviruses. While VZV gK has not been characterized, and its role in viral replication is unknown, homologs of VZV gK in herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV) have been well studied. To identify the VZV ORF5 gene product, we raised a polyclonal antibody against a fusion protein of ORF5 codons 25 to 122 with glutathione S-transferase and used it to study the protein in infected cells. A 40,000-molecular-weight protein was detected in cell-free virus by Western blotting. In immunogold electron microscopic studies, VZV gK was in enveloped virions and was evenly distributed in the cytoplasm in infected cells. To determine the function of VZV gK in virus growth, a series of gK deletion mutants were constructed with VZV cosmid DNA derived from the Oka strain. Full and partial deletions in gK prevented viral replication when the gK mutant cosmids were transfected into melanoma cells. Insertion of the HSV-1 (KOS) gK gene into the endogenous VZV gK site did not compensate for the deletion of VZV gK. The replacement of VZV gK at a nonnative AvrII site in the VZV genome restored the phenotypic characteristics of intact recombinant Oka (rOka) virus. Moreover, gK complementing cells transfected with a full gK deletion mutant exhibited viral plaques indistinguishable from those of rOka. Our results are consistent with the studies of gK proteins of HSV-1 and PRV showing that gK is indispensable for viral replication.

Amino Acid Sequence↗
PubMed1990

Multiple origins of replication in the dihydrofolate reductase amplicons of a methotrexate-resistant chinese hamster cell line.

We recently showed that replication initiates in the early S period at two closely spaced zones in the 240-kilobase (kb) dihydrofolate reductase (DHFR) amplicon of the methotrexate-resistant Chinese hamster ovary cell line CHOC 400. Both of these initiation loci (ori-beta and ori-gamma) have previously been cloned in a recombinant cosmid. In this study, we identified a third early-firing initiation locus (ori-alpha) in the much larger DHFR amplicon of the independently isolated methotrexate-resistant Chinese hamster cell line DC3F-A3/4K (A3/4K). We describe the molecular cloning of this newly identified locus and demonstrate by chromosomal walking that ori-alpha lies approximately 240 kb upstream from ori-beta. Using overlapping cosmid clones for more than 450 kb of DNA sequence from this region of the DHFR domain, we have monitored the replication pattern of the amplicons in synchronized A3/4K cells. These studies suggest that ori-alpha, ori-beta, and ori-gamma are the only early-firing initiation sites in this 450-kb sequence. In addition, we have been able to roughly localize the termini between ori-alpha and ori-beta and between ori-alpha and the next origin in the 5' direction. Thus, we have now isolated the equivalent of three early-firing replicons (including their origins) from a well-characterized chromosomal domain. With these tools, it should be possible to determine those properties that are shared by the origins and termini of different replicons and which are therefore likely to be functionally significant.

Animals↗
PubMed1990

Transcription initiation sites and structural organization of the extreme 5' region of the rat neural cell adhesion molecule gene.

Through analysis of rat genomic cosmid clones, the 5'-most exon of the rat neural cell adhesion molecule (NCAM) gene was identified. This exon, here named exon 0, contained the entire 5' untranslated region and the N-terminal signal sequence of the polypeptide. Exon 0 was isolated from a 1.6-kilobase (kb) EcoRI-HindIII fragment of rat genomic cosmid clone 9 which was 35 kb in length. This fragment was sequenced and found to contain approximately 940 base pairs (bp) of 5'-flanking sequence, exon 0, which was approximately 245 bp in length, and approximately 400 bp of the following intron 0. By using information derived from this fragment and the pR18 rat NCAM cDNA, the transcription initiation sites were determined with two assays. Both primer extensions and nuclease S1 protection assays of postnatal day 7 rat brain RNA identified seven initiation sites within a single 10-bp region at positions -195 to -186 relative to the translation start site. An additional minor site was found at position -329. In the immediate 5' region, no consensus TATA or CCAAT sequences were found. Potential regulatory elements within this region include Sp1 consensus binding sites and also a 178-bp homopurine-homopyrimidine sequence containing several mirror repeats. NCAM has multiple transcripts which are regulated in a developmental and tissue-specific fashion. To determine whether these transcripts are initiated at the same sites, transcription initiation sites were analyzed in postnatal day 7 and adult rat brain and also in cultured cell lines of neuronal, glial, and muscle phenotypes. These tissues and cells exhibited distinct NCAM transcript populations in Northern (RNA) dot blot analysis. In all cases similar transcription start sites were found, suggesting that all major NCAM transcripts have similar or identical initiation sites. These results provide essential information to begin analysis of NCAM regulation in different tissues and during development.

Aging↗
PubMed1998

Substitution of the human beta-spectrin promoter for the human agamma-globin promoter prevents silencing of a linked human beta-globin gene in transgenic mice.

During development, changes occur in both the sites of erythropoiesis and the globin genes expressed at each developmental stage. Previous work has shown that high-level expression of human beta-like globin genes in transgenic mice requires the presence of the locus control region (LCR). Models of hemoglobin switching propose that the LCR and/or stage-specific elements interact with globin gene sequences to activate specific genes in erythroid cells. To test these models, we generated transgenic mice which contain the human Agamma-globin gene linked to a 576-bp fragment containing the human beta-spectrin promoter. In these mice, the beta-spectrin Agamma-globin (betasp/Agamma) transgene was expressed at high levels in erythroid cells throughout development. Transgenic mice containing a 40-kb cosmid construct with the micro-LCR, betasp/Agamma-, psibeta-, delta-, and beta-globin genes showed no developmental switching and expressed both human gamma- and beta-globin mRNAs in erythroid cells throughout development. Mice containing control cosmids with the Agamma-globin gene promoter showed developmental switching and expressed Agamma-globin mRNA in yolk sac and fetal liver erythroid cells and beta-globin mRNA in fetal liver and adult erythroid cells. Our results suggest that replacement of the gamma-globin promoter with the beta-spectrin promoter allows the expression of the beta-globin gene. We conclude that the gamma-globin promoter is necessary and sufficient to suppress the expression of the beta-globin gene in yolk sac erythroid cells.

Animals↗
PubMed1999

Cell differentiation during sexual development of the fungus Sordaria macrospora requires ATP citrate lyase activity.

During sexual development, mycelial cells from most filamentous fungi differentiate into typical fruiting bodies. Here, we describe the isolation and characterization of the Sordaria macrospora developmental mutant per5, which exhibits a sterile phenotype with defects in fruiting body maturation. Cytological investigations revealed that the mutant strain forms only ascus precursors without any mature spores. Using an indexed cosmid library, we were able to complement the mutant to fertility by DNA-mediated transformation. A single cosmid clone, carrying a 3.5-kb region able to complement the mutant phenotype, has been identified. Sequencing of the 3.5-kb region revealed an open reading frame of 2.1 kb interrupted by a 66-bp intron. The predicted polypeptide (674 amino acids) shows significant homology to eukaryotic ATP citrate lyases (ACLs), with 62 to 65% amino acid identity, and the gene was named acl1. The molecular mass of the S. macrospora ACL1 polypeptide is 73 kDa, as was verified by Western blot analysis with a hemagglutinin (HA) epitope-tagged ACL1 polypeptide. Immunological in situ detection of the HA-tagged polypeptide demonstrated that ACL is located within the cytosol. Sequencing of the mutant acl1 gene revealed a 1-nucleotide transition within the coding region, resulting in an amino acid substitution within the predicted polypeptide. Further evidence that ACL1 is essential for fruiting body maturation comes from experiments in which truncated and mutated versions of the acl1 gene were used for transformation. None of these copies was able to reconstitute the fertile phenotype in transformed per5 recipient strains. ACLs are usually involved in the formation of cytosolic acetyl coenzyme A (acetyl-CoA), which is used for the biosynthesis of fatty acids and sterols. Protein extracts from the mutant strain showed a drastic reduction in enzymatic activity compared to values obtained from the wild-type strain. Investigation of the time course of ACL expression suggests that ACL is specifically induced at the beginning of the sexual cycle and produces acetyl-CoA, which most probably is a prerequisite for fruiting body formation during later stages of sexual development. We discuss the contribution of ACL activity to the life cycle of S. macrospora.

ATP Citrate (pro-S)-Lyase↗
PubMed1987

Characterization of an episome produced in hamster cells that amplify a transfected CAD gene at high frequency: functional evidence for a mammalian replication origin.

In a previous study (G. M. Wahl, B. Robert de Saint Vincent, and M. L. De Rose, Nature (London) 307:516-520, 1984), we used gene transfer of a CAD cosmid to demonstrate that gene position profoundly affects amplification frequency. One transformant, T5, amplified the donated CAD genes at a frequency at least 100-fold higher than did the other transformants analyzed. The CAD genes in T5 and two drug-resistant derivatives were chromosomally located. In this report, we show that a subclone of T5 gives rise to an extrachromosomal molecule (CAD episome) containing the donated CAD genes. Gel electrophoresis indicated that the CAD episome is approximately 250 to 300 kilobase pairs, and a variety of methods showed that it is a covalently closed circle. We show that the CAD episome replicates semiconservatively and approximately once per cell cycle. Since the CAD cosmid, which comprises most of the CAD episome, does not replicate autonomously when transfected into cells, our results indicate that either the process which generated the episome resulted in a cellular origin of DNA replication being linked to the CAD sequences or specific rearrangements within the episome generated a functional origin. The implications of these results for mechanisms of gene amplification and the genesis of minute chromosomes are discussed.

Animals↗
PubMed1987

Molecular cloning of fused, a gene required for normal segmentation in the Drosophila melanogaster embryo.

Using the chromosomal walk technique, we isolated recombinant lambda bacteriophage and cosmid clones spanning 250 kilobases (kb) in the 17C-D region of the X chromosome of Drosophila melanogaster. This region was known to contain the segment polarity gene fused. Several lethal fused mutations were used to define more precisely the localization of this locus. Southern analysis of genomic DNA revealed that all of them were relatively large deficiencies, the smallest one being 40 kb long. None of the 12 viable fused mutations examined possessed detectable alterations. We isolated a cosmid containing an insertion covering the entire smallest fused deletion (40 kb). We injected this DNA into fused mutant embryos and obtained a partial phenotypic rescue of the embryonic pattern, indicating that this region contained all the sequences necessary for the embryonic expression of the fu+ gene. Within this DNA, a subclone of 14 kb codes for poly(A)+ RNAs of 3.5, 2.5, 1.6, and 1.3 kb detected in embryos from various developmental stages as well as in adults. All these transcripts showed the same developmental expression. This transcribed region was injected into fused mutant embryos, and once again we obtained a partial rescue of the embryonic phenotype, confirming that this region contained at least the fused gene.

Animals↗
PubMed1988

A fetal globin gene mutation in A gamma nondeletion hereditary persistence of fetal hemoglobin increases promoter strength in a nonerythroid cell.

Single base substitutions have been identified in the promoter regions of A gamma-globin genes from individuals with certain types of nondeletion A gamma hereditary persistence of fetal hemoglobin (HPFH). The presence of these mutations is closely associated with the A gamma HPFH phenotype, but proof that they are the nondeletion HPFH determinants is lacking. To test directly whether these base substitutions can result in an increase in A gamma-globin gene transcription, we studied cosmid clones containing the G gamma- through beta-globin gene regions from individuals with Greek-type (G-to-A base substitution at -117) and Chinese-type (C-to-T base substitution at -196) A gamma HPFH in a transient expression assay. When tested as part of a cosmid clone, the Greek HPFH A gamma-globin gene consistently produced about 1.4 times as much RNA as the wild-type A gamma-globin gene when standardized against RNA transcribed from the G gamma genes in cis. The relative strengths of the normal and HPFH A gamma-globin gene promoters were also compared in transient expression assays with plasmids containing the A gamma-globin genes. Pseudo-wild-type A gamma-globin genes containing a short, transcriptionally neutral deletion were used so that two A gamma-globin genes that differed in their promoter sequences could be compared in the same transfection. The plasmid transient expression results indicated a 1.3- to 1.4-fold increase in steady-state RNA levels from the Greek-type A gamma HPFH promoter compared with the wild-type A gamma promoter, while no difference was documented between the Chinese-type A gamma HPFH promoter and the wild-type A gamma promoter.

Cell Line↗
PubMed1994

Isolation of a new species-specific repetitive sequence from Thinopyrum elongatum and its use in the studies of alien translocations.

A new repetitive sequence that is extremely abundant and well dispersed in the Thinopyrum elongatum genome but present in low-copy number in wheat (Triticum aestivum L.) has been isolated. This repeat and a Th. elongatum repeat isolated in another laboratory were used to identify cosmid genomic clones containing the repeats and, thus, putatively located on a Th. elongatum/T. aestivum translocation arm. Most of the selected cosmids contained single- or low-copy sequences, making them potentially useful in mapping studies. The repeats were used in deletion mapping to deduce gene order of three genes located on the Th. elongatum translocation arm. In situ hybridization studies suggested that this newly identified Th. elongatum repeat is well dispersed throughout the Thinopyrum genome but present at only one location in wheat. This raises some interesting questions about the role of such repetitive elements in the evolution of grass species.

Base Sequence↗
PubMed1996

A 150-megadalton plasmid in Rhizobium etli strain TAL182 contains genes for nodulation competitiveness on Phaseolus vulgaris L.

Rhizobium etli TAL182, a competitive strain for the nodulation of Phaseolus beans, occupied more than 99% of the nodules when co-inoculated in various proportions with Rhizobium TAL1145 or Rhizobium tropici CIAT899. Two overlapping cosmid clones, pUHR68 and pUHR69, containing genes for nodulation competitiveness from TAL182, were isolated by functional complementation of strain TAL1145. Using one of these cosmid clones, we constructed two Tn5-insertion mutants of TAL182 defective in nodulation competitiveness. The Tn5 insertions in both mutants were localized in identical positions within a 4.6-kb HindIII fragment. One mutant, RUH120, was complemented for nodulation competitiveness by this HindIII fragment. The cloned DNA in pUHR68 is a part of a plasmid, 150 MDa in size, in TAL182 and does not show homology with TAL1145 genomic DNA. The 4.6-kb HindIII fragment contains a gene(s) required for nodulation competitiveness on beans, which is present only in some R. etli strains and absent in other Rhizobium spp.

Cosmids↗
PubMed1995

Assignment of Tp53 and Tk1 to chromosome 13 in Peromyscus by fluorescence in situ hybridization.

Mus domesticus DNA probes for the tumor suppressor protein-53 (Tp53) and thymidine kinase-1 (Tk1) genetic loci were used to identify clones representing these loci in a Peromyscus leucopus (white-footed mouse) cosmid library. The cosmid-derived homologous probes were biotinylated and hybridized to P. maniculatus (deer mouse) chromosomes. Probes for both genes hybridized to the chromosome 13 pair identified by prior G-banding. Deer mouse chromosome 13 shares a region of homology with mouse chromosome 11, rat chromosome 10 and human chromosome 17.

Animals↗
PubMed1995

Assignment of the glucose transporter 1 gene (SLC2A1) to swine chromosome 6q34-->qter.

A cosmid clone containing the glucose transporter gene (SLC2A1) was selected from a cosmid library of swine genomic fragments by colony hybridization using the cDNA of swine glucose transporter as a probe. The genomic fragment thus cloned was partially sequenced, and the sequence was compared first with the sequence of the cDNA and then with the reported rat genomic sequence. The sequence homology and exon/intron structure indicated that the genomic fragment contained at least part of SLC2A1. The fragment, together with vector DNA, was then labeled by biotin and used as a probe for in situ hybridization on swine chromosome spreads. Hybridization was visualized by the FITC-labeled streptavidin/biotinylated anti-streptavidin system together with the R-band pattern of chromosomes. The fluorescence signals revealed that SLC2A1 is localized on swine chromosome 6q34-->qter.

Animals↗
PubMed1996

+P5 (D1S3309E), a novel target binding site for the Wilms' tumour suppressor 1 (WT1) gene, maps to human chromosome 1q21-->22.

The Wilms' tumor suppressor 1 gene (WT1) encodes a zinc finger transcription factor critical for normal urogenital development. We have previously isolated a DNA fragment, +P5 (D1S3309E), to which all WT1 protein isoforms bind. Using PCR of a human x rodent somatic cell hybrid mapping panel, together with two-color fluorescence in situ hybridisation of +P5-containing cosmids and previously localised human chromosome 1q cosmids, we have mapped the +P5 fragment to chromosome 1q21-->q22.

Animals↗
PubMed1996

Mapping of 29 YAC clones and identification of 3 YACs spanning the translocation t(3;8)(p14.2;q24.1) breakpoint at 8q24.1 in hereditary renal cell carcinoma.

The constitutional balanced translocation (3;8)(p14.2:q24.1) has been described in a family in which a arge number of individuals developed renal cell carcinoma RCC) at an early age. The translocation event in which genes from the 3p14.2 and the 8q24.1 sites are brought in close proximity is considered a critical, initial step for the development and progression of RCC. Even though the 3p14.2 breakpoint region has been cloned, a gene has not yet been identified, which may be responsible for either the initiation or progression of hereditary RCC. As a crucial step toward cloning the 3;8 breakpoint at the 8q24.1 site, we have mapped a series of YACs which surround this region by fluorescence in situ hybridization (FISH). Three YACs have been identified that span the 8q24.1 breakpoint region. One of these YACs is approximately 180 kb in length, and has been used to initiate construction of a high resolution cosmid contig. Several cosmids have been isolated which have been positioned in relation to the 8q24.1 breakpoint region. In addition, we have positioned 26 other YACs in relation to the 3;8 translocation breakpoint. These results provide a basis for the isolation of genes surrounding 3;8 RCC translocation breakpoint region at 8q24.1.

Carcinoma, Renal Cell↗