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PubMed1984

The rep mutation. VII. Cloning and analysis of the functional rep gene of Escherichia coli K-12.

The rep gene of Escherichia coli was isolated on a 6-kb PvuII fragment of plasmid pLC44-7 DNA from the Clarke-Carbon collection and cloned into pSC101 (to form pHBH8) and pBR322 (to form pHBH30). The plasmids pHBH8 and pHBH30 were found to complement all rep mutations tested. The functional rep gene and its promoter were mapped to a 3.2-kb XhoI-BalI fragment on the basis of complementation data with deletion and insertion derivatives of the two plasmids; subcloning of various restriction fragments confirmed the assignment. EcoRI, HindIII, and HpaI restriction sites were found to reside within that region of the DNA required for expression of the rep function. A coupled in vitro transcription-translation system was used to show that only those plasmids containing a functional rep gene encoded a protein of about Mr 67 000 (the Mr of the rep protein). No plasmids were found that complemented only the A or B classes of rep mutants (which differ in their ability to support the growth of P2 and M13 phages). This result suggests that rep-A and rep-B are alleles of the same structural gene.

Adenosine Triphosphatases↗
PubMed1989

In vivo transfection of murine lungs with a functioning prokaryotic gene using a liposome vehicle.

The authors report successful in vivo transfection of lungs of mice with a functioning prokaryotic gene encoding the intracellular enzyme, chloramphenicol acetyltransferase (CAT). Transfection was accomplished by injecting a plasmid containing the coding region for CAT driven by the SV40 early promoter (pSV2CAT) complexed to specially synthesized cationic liposomes. Intravenous or intratracheal injection of DNA-liposomes resulted in expression of the CAT gene in the lungs, persisting for at least a week, with little enzyme activity detectable in systemic organs. This method should permit either transient or stable in vivo transfection of the lungs with a gene encoding any protein of interest, providing a powerful experimental tool and potentially a novel and broadly applicable clinical therapeutic technique.

Acetylation↗
PubMed2004

Identification of a second flagellin gene and functional characterization of a sigma70-like promoter upstream of a Leptospira borgpetersenii flaB gene.

Leptospira borgpetersenii, one of the causative agents of leptospirosis in both animals and humans, is a bacterial pathogen with characteristic motility that is mediated by the rotation of two periplasmic flagella (PF). The flaB gene coding for a core polypeptide subunit of PF was previously characterized by sequence analysis of its open reading frame (ORF) (M. Lin, J Biochem Mol Biol Biophys 2:181-187, 1999). The present study was undertaken to isolate and clone the uncharacterized sequence upstream of the flaB gene by using a PCR-based genome walking procedure. This has resulted in a 1470-bp genomic DNA sequence in which an 846-bp ORF coding for a 281-amino acid polypeptide (31.3 kDa) is identified 455 bp upstream from the flaB start codon. The encoded protein exhibits 72% amino acid identity to the deduced FlaB protein sequence of L. borgpetersenii and a high degree of sequence homology to the FlaB proteins of other spirochaetes. This has demonstrated for the first time that a second flaB gene homolog is present in a Leptospira species. The newly identified gene is designated flaB1, and the previously cloned flaB renamed flaB2. Within the intergenic sequence between flaB1 and flaB2, a potential stem-loop structure (12-bp inverted repeats) was identified 25 bp downstream of the flaB1 stop codon; this could serve as a transcription terminator for the flaB1 mRNA. Three E. coli-like promoter regions (I, II, and III) for binding Esigma(70), a regulatory sequence uncommonly found in flagellar genes, were predicted upstream of the flaB2 ORF. Only promoter region II contains a promoter that is functional in E. coli, as revealed at phenotypic and transcriptional levels by its capability of directing the expression of the chloramphenicol acetyltransferase (CAT) gene in the promoter probe vector pKK232-8. These observations may suggest that flaB1 and flaB2 are transcribed separately and do not form a transcriptional operon controlled by a single promoter.

Bacterial Proteins↗
PubMed2006

Nanog retrotransposed genes with functionally conserved open reading frames.

The Nanog gene plays a key role in the pluripotency of early embryonic cells in vitro and in vivo. In this article retrotransposed copies of Nanog, termed NanogPc and NanogPd, are identified on mouse Chromosomes 4 and 7, respectively. In contrast to the two previously characterized mouse Nanog retrogenes that contain multiple frameshifts and point mutations, NanogPc and NanogPd are 98% identical to NANOG within the open reading frame and encode proteins with activity in an embryonic stem cell self-renewal assay. Mutations common to all four retrotransposed genes but distinct from Nanog suggest divergence from a common progenitor that appears likely to be Nanog because transcripts derived from Nanog but not from the retrogenes are detected in germ-line cells. The possibility that expression of Nanog could be erroneously attributed to novel cellular sources is suggested by the high homology among Nanog, NanogPc, and NanogPd. Analysis of distinct Mus species suggests that NanogPc and NanogPd arose between divergence of M. caroli and M. spretus and indicates that Nanog retrotransposition events continue to occur at a high frequency, a property likely to extend to other germ-line transcripts.

Amino Acid Sequence↗
PubMed1996

Identification of a Stat gene that functions in Drosophila development.

A Drosophila Stat gene (D-Stat) with a zygotic segmental expression pattern was identified. This protein becomes phosphorylated on Tyr-704 when coexpressed in Schneider cells with a Drosophila janus kinase (JAK), Hopscotch (HOP). The phosphorylated protein binds specifically to the consensus sequence TTCCCGGAA. Suppressor mutations of hopTum-I, a dominant hyperactive allele of hop whose phenotype is hematocyte overproduction and tumor formation, were selected. One of these mutants, statHJ, mapped to the same chromosomal region (92E) as does D-Stat, had an incompletely penetrant pair rule phenotype, and exhibited aberrant expression of the pair rule gene even skipped (eve) at the cellular blastoderm stage. Two D-STAT-binding sites were identified within the eve stripe 3 enhancer region. Mutations in either of the STAT-binding sites greatly decreased the stripe 3 expression in transgenic flies. Clearly, the JAK-STAT pathway is connected to Drosophila early development.

Amino Acid Sequence↗
PubMed1994

The 50-kDa primase subunit of Drosophila melanogaster DNA polymerase alpha. Molecular characterization of the gene and functional analysis of the overexpressed protein.

The gene encoding the 50-kDa subunit of Drosophila melanogaster DNA polymerase alpha has been cloned. A comparison of the predicted polypeptide sequence of the Drosophila protein with the equivalent subunits from mouse and yeast suggests that they are closely related and defines three conserved regions which are likely to be important for enzyme activity. The expression patterns of both the 50-kDa protein and its transcript (a single RNA message of 1.6 kilobases) throughout development are consistent with a role of the protein in DNA replication. When overexpressed and purified the 50-kDa subunit displays DNA primase activity. The products of the reaction, mainly oligoribonucleotides 12-14 nucleotides in length, plus dimers and some trimers, are similar to those synthesized by either the intact DNA polymerase alpha, or the biochemically isolated primase heterodimer. The isolated primase also shows similar sensitivity to antibodies, magnesium and monovalent cations, and the same nucleotide requirements as complexed forms of the primase. The isolated subunit, however, is more thermally labile, suggesting a role for the additional subunits in DNA polymerase alpha in stabilizing the primase activity of the 50-kDa primase subunit.

Amino Acid Sequence↗
PubMed1998

Isolation of a novel heat shock protein 70-like gene, pss1+ of Schizosaccharomyces pombe homologous to hsp110/SSE subfamily.

A novel heat shock protein 70 (HSP70) gene, pss1+, of fission yeast, Schizosaccharomyces pombe (S. pombe), has been isolated as a multicopy suppressor of a synthetic lethal mutant of ras1+, which shows severe retardation of growth and aggregation phenotype when the ras1 gene function is absent. The pss1+ gene functionally complements the growth defect of the mutant. Sequence analysis revealed that pss1+ encodes an open reading frame (ORF) of 730amino acids that is homologous to the HSP70 family proteins. The Pss1 has high homology to the Saccharomyces cerevisiae (S. cerevisiae) heat shock protein Sse1p/Msi3p (43% identity) that belongs to the HSP110/SSE subfamily of HSP70. The consensus nucleotide sequence of the heat shock element (HSE) was found in the upstream region of pss1+ gene. The transcript level of pss1+ was moderately abundant during steady-state growth at 25 degrees C and increased a few-fold upon shifting to 42 degrees C. Furthermore, transcription of pss1+ increased in nitrogen-starved conditions. Disruption of the pss1+ gene confers a temperature-sensitive growth phenotype and unexpectedly causes the increase in thermotolerance in S. pombe.

Amino Acid Sequence↗
PubMed1983

Expression of adenovirus E1a and E1b gene products and the Escherichia coli XGPRT gene in KB cells.

The recombinant plasmid pSV2-gpt, which contains the Escherichia coli XGPRT gene under the control of a simian virus 40 early promoter, was modified to contain the type 2 adenovirus (Ad2) XhoI-C (0 to 15.5 map units) restriction endonuclease fragment. Plasmid (pLB206) DNA was introduced into human KB cells by Ca2+-mediated DNA transfection, and transformants were selected in medium containing xanthine, aminopterin, and mycophenolic acid, as a consequence of expression of the dominant, selectable XGPRT gene. A series of 13 gpt+ cell lines were isolated and tested for their ability to complement Ad5 deletion mutants in E1a (H5dl312) and E1b (H5dl315). Four classes of gpt+ KB cell lines were identified, including clones constitutively expressing both E1a and E1b, only E1a, or only E1b or not expressing either E1a or E1b. DNA and RNA filter transfer hybridization analysis substantiated the conclusions that those cell lines capable of complementing viral host range mutants contained the appropriate viral DNA sequences and cytoplasmic polyadenylated RNA species. DNA filter transfer hybridization studies also revealed that the transfected vector DNA was stably integrated into chromosomal DNA in the KB transformants and the number of integrated sites ranged from 1 to 3. The gpt+ KB cell line that only expressed E1b gene functions only contained viral E1b gene sequences; those cell lines that expressed neither E1a nor E1b gene function contained only small or no regions of Ad2 DNA. When weaned off the selective medium, transformed KB cell lines stably maintained their inserted DNA in the absence of selective pressure and could easily be adapted to growth in suspension culture.

Adenoviruses, Human↗
PubMed2006

Caenorhabditis elegans has two genes encoding functional d-aspartate oxidases.

Four cDNA clones that were annotated in the database as encoding d-amino acid oxidase (DAAO) or d-aspartate oxidase (DASPO) were isolated by RT-PCR from Caenorhabditis elegans RNA. The proteins (Y69Ap, C47Ap, F18Ep, and F20Hp) encoded by the cloned cDNAs were expressed in Escherichia coli as recombinant proteins with an N-terminal His-tag. All proteins except F20Hp were recovered in the soluble fractions. The recombinant Y69Ap has functional DAAO activity, as it can deaminate neutral and basic d-amino acids, whereas the recombinants C47Ap and F18Ep have functional DASPO activities, as they can deaminate acidic d-amino acids. Additional experiments using purified recombinant proteins revealed that Y69Ap deaminates d-Arg more efficiently than d-Ala and d-Met, and that C47Ap and F18Ep show distinct kinetic properties against d-Asp, d-Glu, and N-methyl-d-Asp. This is the first time that cDNA cloning of invertebrate DAAO and DASPO genes has been reported. In addition, our study reveals for the first time that C. elegans has at least two genes encoding functional DASPOs and one gene encoding DAAO, although it had previously been thought that organisms only bear one copy each of these genes. The two C. elegans DASPOs differ in their substrate specificities and possibly also in their subcellular localization.

Amino Acid Sequence↗
PubMed2005

Clues to function in gene deserts.

Recent work by Ivan Ovcharenko and colleagues has shed new light on the functional importance of gene deserts. They demonstrate that sequence conservation levels separate gene deserts into stable (more conserved) and variable classes. Both classes exhibit characteristics suggestive of function. The stable deserts in particular show features suggesting a role in the complex regulation of core vertebrate genes.

DNA, Intergenic↗
PubMed1991

Cloning and structural analysis of two distinct families of ovine interferon-alpha genes encoding functional class II and trophoblast (oTP) alpha-interferons.

Ovine trophoblast protein (oTP) is a polypeptide secreted by ovine trophectoderm from day 11 to 21, which plays a key role in maternal recognition of pregnancy. Structural analyses established that oTP shares extensive homology with class II alpha-interferon (IFN-alpha II) subfamily. Previous screening of an ovine genomic DNA library probed with an oTP cDNA incidently resulted in the isolation of a functional IFN-alpha II gene and two relevant pseudogenes, as shown by sequence analysis and study of expression in eukaryotic COS cells. The expected oTP gene together with a cognate pseudogene was successfully isolated from the series of clones selected from another genomic library probed with the oTP cDNA, using two specific oligonucleotides, each one complementary to a region of oTP cDNA with little homology with the IFN-alpha II gene and related pseudogenes. Southern blotting of ovine genomic DNA indicated the existence of at least five trophoblast IFN-alpha genes or pseudogenes. Nucleotide sequence comparisons showed that the oTP gene exhibits a higher homology (90%) with bovine trophoblast IFN gene (Stewart et al. (1990) J. Mol. Endocrinol. 4, 275-282) than with oIFN-alpha II gene (70%), thus providing evidence that embryonic IFNs constitute a distinct subfamily of IFN-alpha s.

Amino Acid Sequence↗
PubMed2006

Kidney-specific gene targeting: insight into thiazolidinedione-induced fluid retention.

Conditional gene targeting technique, which is based on the use of Cre-loxP or Flp/FRT systems, has been increasingly used to study gene function of a particular cell type in vivo. The introduction of this technique to the kidney field is relatively recent but has already provided important insights into physiological or pathological functions of a number of genes in the kidney. This technique has recently been used to inactivate the peroxisome proliferator-activated receptor subtype gamma in the collecting duct, which leads to remarkable blockade of body weight gains and plasma volume expansion associated with thiazolidinediones. This finding not only helps understand pharmacology of the novel class of antidiabetic drugs, but also uncovers an important role of peroxisome proliferator-activated receptor subtype gamma in regulation of distal nephron fluid reabsorption. The present review represents an example for the use of the modern technique to address complex clinical problems. It is anticipated that over next few years this technique will be used by an increasing number of investigators for studying gene function in the kidney.

Animals↗
PubMed2005

The structure and population genetics of the breakpoints associated with the cosmopolitan chromosomal inversion In(3R)Payne in Drosophila melanogaster.

We report here the breakpoint structure and sequences of the Drosophila melanogaster cosmopolitan chromosomal inversion In(3R)P. Combining in situ hybridization to polytene chromosomes and long-range PCR, we have identified and sequenced the distal and proximal breakpoints. The breakpoints are not simple cut-and-paste structures; gene fragments and small duplications of DNA are associated with both breaks. The distal breakpoint breaks the tolkin (tok) gene and the proximal breakpoint breaks CG31279 and the tolloid (tld) gene. Functional copies of all three genes are found at the opposite breakpoints. We sequenced a representative sample of standard (St) and In(3R)P karyotypes for a 2-kb portion of the tok gene, as well as the same 2 kb from the pseudogene tok fragment found at the distal breakpoint of In(3R)P chromosomes. The tok gene in St arrangements possesses levels of polymorphism typical of D. melanogaster genes. The functional tok gene associated with In(3R)P shows little polymorphism. Numerous single-base changes, as well as deletions and duplications, are associated with the truncated copy of tok. The overall pattern of polymorphism is consistent with a recent origin of In(3R)P, on the order of Ne generations. The identification of these breakpoint sequences permits a simple PCR-based screen for In(3R)P.

Animals↗
PubMed1988

Separate genes encode functionally equivalent ADP/ATP carrier proteins in Saccharomyces cerevisiae. Isolation and analysis of AAC2.

Genetic and biochemical analysis of Saccharomyces cerevisiae containing a disruption of the nuclear gene (AAC1) encoding the mitochondrial ADP/ATP carrier has revealed a second gene for this protein. The second gene, designated AAC2, has been isolated by genetic complementation and sequenced. AAC2 contains a 954-base pair open reading frame coding for a protein of 318 amino acids which is highly homologous to the AAC1 gene product except that it is nine amino acids longer at the NH2 terminus. The two yeast genes are highly conserved at the level of DNA and protein and share identity with the ADP/ATP carriers from other organisms. Both genes complement an ADP/ATP carrier defect (op1 or pet9). However, the newly isolated gene AAC2 need be present only in one or two copies while the previously isolated AAC1 gene must be present in multiple copies to support growth dependent on a functional carrier protein. This gene dosage-dependent complementation combined with the high degree of conservation suggest that these two functionally equivalent genes may be differentially expressed.

Amino Acid Sequence↗
PubMed2003

Using yeast to place human genes in functional categories.

The availability of the draft sequence of the human genome has created a pressing need to assign functions to each of the 35,000 or so genes that it defines. One useful approach for this purpose is to use model organisms for both bioinformatic and functional comparisons. We have developed a complementation system, based on the model eukaryote Saccharomyces cerevisiae, to clone human cDNAs that can functionally complement yeast essential genes. The system employs two regulatable promoters. One promoter, tetO (determining doxycycline-repressible expression), is used to control essential S. cerevisiae genes. The other, pMET3 (which is switched off in the presence of methionine), is employed to regulate the expression of mammalian cDNAs in yeast. We have demonstrated that this system is effective for both individual cDNA clones and for cDNA libraries, permitting the direct selection of functionally complementing clones. Three human cDNA libraries have been constructed and screened for clones that can complement specific essential yeast genes whose expression is switched off by the addition of doxycycline to the culture medium. The validity of each complementation was checked by showing that the yeast cells stop their growth in the presence of doxycycline and methionine, which represses the expression of the yeast and mammalian coding sequence, respectively. Using this system, we have screened 25 tetO replacement strains and succeeded in isolating human cDNAs complementing six essential yeast genes. In this way, we have uncovered a novel human ubiquitin-conjugating enzyme, have isolated a human cDNA clone that may function as a signal peptidase and have demonstrated that the functional segment of the human Psmd12 proteosome sub-unit contains a PINT domain.

Cerebellum↗
PubMed2004

Developmental and cell-specific expression of ZWICHEL is regulated by the intron and exon sequences of its gene.

Functional studies with ZWICHEL ( ZWI ), which encodes a Ca(2+)-calmodulin-regulated kinesin, have shown its involvement in trichome morphogenesis and cell division. To identify regulatory regions that control the ZWI expression pattern, we generated transgenic Arabidopsis plants with a GUS reporter driven by different lengths of the ZWI gene 5' region alone or 5' and 3' regions together. The 5' fusions contain varying lengths of the coding and non-coding regions of beta - HYDROXYISOBUTYRYL-CoA HYDROLASE 1 ( CHY1 ), which is upstream of ZWI, and a 162 bp intergenic region. In transgenic plants with 5' 460::GUS, GUS activity was observed primarily in the root hairs whereas transgenic plants with an additional 5' 266 bp region from the CHY1 gene (5' 726::GUS) showed strong GUS accumulation in the entire root including root hairs and root tip, calli and at various developmental stages in trichomes and pollen. However, very little GUS accumulation was detected in roots of dark-grown or root tips of cold-treated seedlings with 5' ZWI constructs. These results were further confirmed by quantifying GUS enzyme activity and transcripts in these seedlings. Calli and pollen transformed with the 5' distal 268 bp fused in antisense orientation to the proximal 460 bp did not show GUS expression. Further, IAA-treated dark-grown seedlings with 726::GUS, but not with 460::GUS, showed high GUS expression in specific regions (outer layer 2a cells) at the base of the lateral roots. The ZWI 3' region (3 kb) did not influence the GUS expression pattern driven by the 5' 726 bp. The absence of CHY1 transcripts in the chy1-2 mutant did not alter either ZWI expression or ZWI-mediated trichome morphogenesis. Thus, our data suggest that the 3' part of the CHY1 gene contains regulatory elements that control ZWI gene expression in dividing cells and other cells that exhibit polarized growth such as root hairs, pollen and trichomes. This is the first evidence that the regulatory regions conferring developmental and cell-specific expression of a gene reside in the introns and exons of its upstream protein-coding gene.

5' Flanking Region↗
PubMed1997

Oligonucleotides as modulators of cancer gene expression.

The delineation of gene function has always been an intensive subject of investigations. Recent advances in the synthesis and chemistry of oligonucleotides have now made these molecules important tools to study and identify gene function and regulation. Modulation of gene expression using oligonucleotides has been targeted at different levels of the cellular machinery. Triplex forming oligonucleotides, as well as peptide nucleic acids, have been used to inhibit gene expression at the level of transcription; after binding of these specific oligonucleotides, conformational change of the DNA's helical structure prevents any further DNA/protein interactions necessary for efficient transcription. Gene regulation can also be achieved by targeting the translation of mRNAs. Antisense oligonucleotides have been used to down-regulate mRNA expression by annealing to specific and determined region of an mRNA, thus inhibiting its translation by the cellular machinery. The exact mechanism of this type of inhibition is still under intense investigation and is thought to be related to the activation of RNase H, a ribonuclease that is widely available that can cleave the RNA/DNA duplex, thus making it inactive. Another well-characterized means of interfering with the translation of mRNAs is the use of ribozymes. Ribozymes are small catalytic RNAs that possess both site specificity and cleavage capability for an mRNA substrate, inhibiting any further protein formation. This review describes how these different oligonucleotides can be used to define gene function and discusses in detail their chemical structure, mechanism of action, advantages and disadvantages, and their applications.

Animals↗
PubMed2006

Electroporation of DNA, RNA, and morpholinos into zebrafish embryos.

The combination of accessible embryology and forward genetic techniques has made zebrafish a powerful model system for the study of vertebrate development. One limitation of genetic analysis is that the study of gene function is usually limited to the first developmental event affected by a gene. In vivo electroporation has recently matured as a method for studying gene function at different developmental time points and in specific regions of the organism. The focal application of current allows macromolecules to be efficiently introduced into a targeted region at any time in the life cycle. Here we describe a rapid protocol by which DNA, RNA and morpholinos can all be precisely electroporated into zebrafish in a temporally and spatially controlled manner. This versatile technique allows gene function to be determined by both gain and loss of function analyses in specific regions at specific times. This is the first report that describes the electroporation of three different molecules into embryonic and larval zebrafish cells.

Animals↗