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At least 127 records · Page 7Linked to original sources

PubMed1997

The evolution of Msx gene function: expression and regulation of a sea urchin Msx class homeobox gene.

Msx- class homeobox genes, characterized by a distinct and highly conserved homeodomain, have been identified in a wide variety of metazoans from vertebrates to coelenterates. Although there is evidence that they participate in inductive tissue interactions that underlie vertebrate organogenesis, including those that pattern the neural crest, there is little information about their function in simple deuterostomes. Both to learn more about the ancient function of Msx genes, and to shed light on the evolution of developmental mechanisms within the lineage that gave rise to vertebrates, we have isolated and characterized Msx genes from ascidians and echinoderms. Here we describe the sequence and expression of a sea urchin (Strongylocentrotus purpouratus) Msx gene whose homeodomain is very similar to that of vertebrate Msx2. This gene, designated SpMsx, is first expressed in blastula stage embryos, apparently in a non-localized manner. Subsequently, during the early phases of gastrulation, SpMsx transcripts are expressed intensely in the invaginating archenteron and secondary mesenchyme, and at reduced levels in the ectoderm. In the latter part of gastrulation, SpMsx transcripts are concentrated in the oral ectoderm and gut, and continue to be expressed at those sites through the remainder of embryonic development. That vertebrate Msx genes are regulated by inductive tissue interactions and growth factors suggested to us that the restriction of SpMsx gene expression to the oral ectoderm and derivatives of the vegetal plate might similarly be regulated by the series of signaling events that pattern these embryonic territories. As a first test of this hypothesis, we examined the influence of exogastrulation and cell-dissociation on SpMsx gene expression. In experimentally-induced exogastrulae, SpMsx transcripts were distributed normally in the oral ectoderm, evaginated gut, and secondary mesenchyme. However, when embryos were dissociated into their component cells, SpMsx transcripts failed to accumulate. These data show that the localization of SpMsx transcripts in gastrulae does not depend on interactions between germ layers, yet the activation and maintenance of SpMsx expression does require cell-cell or cell-matrix interactions.

Animals↗

PubMed1980

DNA methylation and gene function.

In most higher organisms, DNA is modified after synthesis by the enzymatic conversion of many cytosine residues to 5-methylcytosine. For several years, control of gene activity by DNA methylation has been recognized as a logically attractive possibility, but experimental support has proved elusive. However, there is now reason to believe, from recent studies, that DNA methylation is a key element in the hierarchy of control mechanisms that govern vertebrate gene function and differentiation.

Animals↗

PubMed1999

Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis.

We describe a genetic mosaic system in Drosophila, in which a dominant repressor of a cell marker is placed in trans to a mutant gene of interest. Mitotic recombination events between homologous chromosomes generate homozygous mutant cells, which are exclusively labeled due to loss of the repressor. Using this system, we are able to visualize axonal projections and dendritic elaboration in large neuroblast clones and single neuron clones with a membrane-targeted GFP marker. This new method allows for the study of gene functions in neuroblast proliferation, axon guidance, and dendritic elaboration in the complex central nervous system. As an example, we show that the short stop gene is required in mushroom body neurons for the extension and guidance of their axons.

Animals↗

PubMed1986

An estrogen-responsive element derived from the 5' flanking region of the Xenopus vitellogenin A2 gene functions in transfected human cells.

In the human breast cancer cell line MCF-7, we observe estrogen induction of the stable transfected Xenopus vitellogenin A2 gene. An estrogen-responsive element (ERE) could be defined by using a vitellogenin-chloramphenicol acetyltransferase hybrid gene in transient transfection experiments. The ERE is located in the 5' flanking region and is able to confer estrogen inducibility to the thymidine kinase gene promoter. By 5' and 3' deletions we have determined a 35 bp sequence sufficient for high stimulation by estradiol. Even 18 bp give a small estrogen response. The 35 bp ERE contains the palindromic sequence 5'GGTCACAGTGACC-3' as an essential element. The fact that the ERE of a frog gene functions in human cells demonstrates that signals and factors involved in the control have been conserved during evolution.

Acetyltransferases↗

PubMed1983

Physical location of a herpes simplex virus type-1 gene function(s) specifically associated with a 10 million-fold increase in HSV neurovirulence.

In this paper we present a technique developed to physically locate the HSV-1 gene(s) which restore neurovirulence to a non-neurovirulent HSV intertypic recombinant described in the preceding report. In brief, tissue culture cells are co-transfected with unit length RE6 DNA and restriction endonuclease fragmented HSV-1 (strain 17 Syn+) DNA. In this way, random recombinations between RE6 and 17 Syn+ are produced. An in vivo enrichment in mouse brains is then employed to select recombinants which have incorporated the HSV-1 gene(s) associated with neurovirulence. In each of five cases where neurovirulent recombinants were isolated by this procedure, restriction enzyme and Southern DNA transfer analysis revealed that HSV-1 information from 0.71 to 0.83 map units had been incorporated into the RE6 genome. Confirmation of the role of this portion of the genome for HSV neurovirulence was obtained by similar cotransfection and in vivo rescue experiments performed with an electrophoretically purified HSV-1 DNA fragment which encompasses this region. Subsequent genome structure analysis of neurovirulent recombinants generated by this procedure revealed that only type-1 information from 0.71 to 0.83 map units had been incorporated into RE6. Thus an HSV-1 gene function(s) which resides in this region of the viral DNA is associated with a 10 million-fold increase in the neurovirulence of the virus. Potential applications of this in vivo selection technique are discussed.

Animals↗

PubMed1999

The maize tha4 gene functions in sec-independent protein transport in chloroplasts and is related to hcf106, tatA, and tatB.

Proteins are translocated across the chloroplast thylakoid membrane by a variety of mechanisms. Some proteins engage a translocation machinery that is derived from the bacterial Sec export system and require an interaction with a chloroplast-localized SecA homologue. Other proteins engage a machinery that is SecA-independent, but requires a transmembrane pH gradient. Recently, a counterpart to this Delta pH mechanism was discovered in bacteria. Genetic studies revealed that one maize protein involved in this mechanism, HCF106, is related in both structure and function to the bacterial tatA and tatB gene products. We describe here the mutant phenotype and molecular cloning of a second maize gene that functions in the Delta pH mechanism. This gene, thylakoid assembly 4 (tha4), is required specifically for the translocation of proteins that engage the Delta pH pathway. The sequence of the tha4 gene product resembles those of the maize hcf106 gene and the bacterial tatA and tatB genes. Sequence comparisons suggest that tha4 more closely resembles tatA, and hcf106 more closely resembles tatB. These findings support the notion that this sec-independent translocation mechanism has been highly conserved during the evolution of eucaryotic organelles from bacterial endosymbionts.

Amino Acid Sequence↗

PubMed1991

Segmental exchange between MHC class I genes in a higher primate: recombination in the gorilla between the ancestor of a human non-functional gene and an A locus gene.

Classical human major histocompatibility complex (MHC) class I molecules are the products of highly diverse gene loci. It has been suggested that segmental exchange may play a role in the generation of diversity at the antigen recognition site of MHC class I molecules. Here we present the cloning, sequencing and expression of two gorilla A locus cDNAs. One of these cDNAs shows remarkable similarity to the non-functional HLA-AR locus gene (5.4-LBF) only in exon 2. The remainder of the cDNA, however, is most closely related to other classical higher primate A locus genes. This suggests that a segmental exchange may have occurred between the ancestor of the non-functional HLA-AR gene and a classical gorilla A locus gene. Furthermore, the recombination event resulting in Gogo-A3 has affected its antigen recognition site. These data, therefore, demonstrate that segmental exchange can generate diversity at the antigen recognition sites of primate MHC class I molecules and suggest that non-functional genes can contribute to the generation of diversity of classical MHC class I genes.

Animals↗

PubMed1989

Molecular cloning and expression of the functional gene encoding the M2 subunit of mouse ribonucleotide reductase: a new dominant marker gene.

Mammalian ribonucleotide reductase consists of two non-identical subunits, proteins M1 and M2. M2-related DNA sequences are present on mouse chromosomes 4, 7, 12 and 13. However, M2-overproducing mouse cells show amplification of a chromosome 12-specific, single 13 kb HindIII fragment, which probably represents the active gene. We have isolated this fragment from parental mouse cell DNA and used it to clone and characterize the functional M2 gene. The 5770 bp transcribed M2 sequence contains ten exons separated by nine 95-917 bp introns. The 501 bp of 5' flanking DNA is G + C rich and contains TTTAAA and CCAAT sequences as well as potential Sp1 binding sites. The M2-related sequence on chromosome 13, which contains only the last six exons and several internal rearrangements, is a pseudogene. Transfection of BALB/3T3 cells with the M2 gene resulted in stable transformants with a 10-fold reduction in sensitivity to hydroxyurea, compared to control cells. This confirmed that the cloned M2 genomic DNA represents the functional gene and conclusively establishes the link between hydroxyurea resistance and M2 expression in mammalian cells. M2 genomic DNA should be a valuable dominant, selectable marker for identifying and isolating stable co-transformants.

Animals↗

PubMed1989

Cellular senescence involves stochastic processes causing loss of expression of differentiated function genes: visualization by in situ hybridization for steroid 17 alpha-hydroxylase in bovine adrenocortical cells.

When grown for long periods in culture, bovine adrenocortical cells lose the expression of a differentiated function gene, steroid 17 alpha-hydroxylase. Previously, we documented a decline in 17 alpha-hydroxylase mRNA with increasing culture passage level after induction with cyclic AMP (P. J. Hornsby et al., 1987, Proc. Natl. Acad. Sci. USA 84, 1580). We used in situ hybridization to investigate the loss of expression of this gene during cellular senescence at an individual cell level. In primary cultures, cells were uniformly positive for hybridization with cDNA for 17 alpha-hydroxylase after cyclic AMP induction. After two passages, cultures comprised a mixture of hybridizing and nonhybridizing cells. Cells appeared either to hybridize at a level comparable to that in primary cultures or to be nonhybridizing. When in situ hybridization was combined with immunofluorescence, cells positive for immunofluorescence were also positive for hybridization. Senescing mass cultures showed decreasing numbers of positive cells, and after 30 passages cultures comprised entirely nonhybridizing cells. Thus, the previously observed decline in overall 17 alpha-hydroxylase mRNA levels results from a decline in the fraction of expressing cells in the culture, and the rate of loss of expressing cells is in agreement with the rate of loss of total 17 alpha-hydroxylase mRNA. Primary clones, even when isolated at an early stage of clonal expansion, had mixtures of subclones of hybridizing and nonhybridizing cells. On recloning, hybridizing subclones usually produced uniformly nonhybridizing sub-subclones. Some subclones within primary clones had a morphology associated with replicative senescence (flattened cells with sparse intercellular contacts), yet had high numbers of hybridizing cells. We conclude that, in both mass and clonal populations, cells initially expressing 17 alpha-hydroxylase rapidly give rise to clones of nonexpressing cells. Such cells are continually derived by a stochastic process from cells originally expressing the gene.

Adrenal Cortex↗

PubMed1998

A point mutation within exon 5 of the WT1 gene of a sporadic unilateral Wilms' tumor alters gene function.

The Wilms' tumor suppressor gene, wt1, encodes a zinc-finger transcription factor, WT1, that represses transcription of a number of growth-promoting genes and inhibits cell growth. The transcripts of wt1 undergo two alternative splicing events, giving rise to four isoforms of mRNA in constant ratios. The first alternative splice introduces an extra exon 5, which encodes 17 amino acid residues inserted between the transcription regulatory domain and the DNA binding domain of WT1. Previously, we demonstrated that the 17-amino acid domain functioned as a transcription repressor when it was fused with the DNA binding domain of WT1. We have now identified a point mutation within exon 5 of wt1 in a sporadic unilateral Wilms' tumor patient. The mutation changes the last of the 17 amino acids from asparagine to serine. The protein isoform of WT1 carrying this mutation exhibited a 2-3-fold lower transcription-repressing activity than wild-type WT1 in transient cotransfection assays. The mutation also decreased growth-inhibiting activity of WT1 in two osteosarcoma cell lines, U2OS and Saos-2. By diminishing transcription-repressing and growth-inhibiting activities of WT1, this naturally occurring mutation within exon 5 of wt1 may disturb the normal function of the protein and lead to the uncontrolled cell growth characteristic of Wilms' tumor.

Alternative Splicing↗

PubMed1978

Evidence for variation in the number of functional gene copies at the AmaR locus in Chinese hamster cell lines.

The hypothesis of functional hemizygosity has been examined for the alpha-amanitin resistant (AmaR, a codominant marker) locus in a series of Chinese hamster cell lines. AmaR mutants were obtained from different cell lines, e.g., CHO, DHW, M3- 1 and CHO-Kl, at similar frequencies. After fractionation of different RNA polymerase activities in the extracts by chromatographic procedures, the sensitivity of the mutant RNA polymerase II towards alpha-amanitin was determined. While all of the RNA polymerase II activity in mutant CHO and CHO-Kl lines became resistant to alpha-amanitin inhibition, only about 50% of the activity is highly resistant in AmaR mutants of CHW and M3- 1 cell lines. The remaining activity in the latter cell lines shows alpha-amanitin sensitivity similar to that seen with the wild-type enzyme. This behaviour is similar to that observed with a 1:1 mixture of resistant and sensitive enzymes from CHO cells. These results, therefore, strongly indicate that while only one functional copy of the gene affected by alpha-amanitin is present in CHO and CHO-Kl cells, two copies of this gene are functional in the CHW and M3-1 cell lines.

Amanitins↗

PubMed1999

Analysis of hexamer and pentamer motifs within a maize database: the presence of motif 'signatures' in functional gene categories.

Several transposable elements (TEs) have been reported in association with genes in maize and other plants. In this study we found, based on statistical analyses of 951 DNA sequences within a maize computer database, that short hexamer and pentamer DNA motifs from the Activator (Ac) and from the Tourist TEs, respectively, were also associated with maize genes. Moreover, these two short hexamer and pentamer TE motifs were nonrandomly and nearly nonrandomly distributed, respectively, with respect to particular biochemical functions of those maize genes. To determine whether this distribution may be unique to TE motifs, or may be more widespread among hexamers/pentamers in general, we similarly studied six additional hexamer or pentamer sequences not derived from TEs. These also showed nonrandom distribution with respect to functional gene categories in the maize database. However, each of the total of eight short sequence motifs we studied differed in its pattern of association with distinct sets of functional gene categories; that is, there was a unique 'signature' for each of the hexamers and pentameters tested. Potential biological hypotheses to explain these findings are discussed.

DNA Transposable Elements↗

PubMed1998

An avian pathogenic Escherichia coli strain produces a hemolysin, the expression of which is dependent on cyclic AMP receptor protein gene function.

An avian pathogenic Escherichia coli strain M1000 showed a clear zone of erythrocyte lysis on sheep blood agar plates. The hemolytic activity was not detected in the culture supernatant nor was any DNA sequence homologous to the E. coli alpha-hemolysin gene detected in the chromosome or plasmid DNA of the strain, indicating that the observed hemolysis was different from alpha-type. To identify the genetic determinant responsible for the hemolysis, we performed random Tn5 insertional mutagenesis and obtained one mutant, named M5005, that totally lacked the hemolytic activity. Cloning and nucleotide sequencing of the region flanking the transposon insertion site in the M5005 chromosome revealed that the transposon was inserted within an open reading frame of the cyclic AMP receptor protein (CRP) gene, which is involved in one of the global regulatory networks of gene expression in E. coli. Nucleotide sequence analysis of the intact crp gene of the strain M1000 showed that the CRP protein of M1000 is 99% identical to that of K-12. Introduction of the intact crp gene on a low copy plasmid into the mutant M5005 restored the hemolytic phenotype, confirming that the mutation site in M5005 was in the crp gene. CRP plays a central role in catabolite repression, the phenomenon by which the synthesis of many enzymes required to metabolize various sugars is repressed in the presence of glucose. When the hemolytic activity of E. coli M1000 grown in the presence of glucose was examined, the hemolysis was totally impaired. These results indicate that the avian pathogenic E. coli strain M1000 produces a hemolysin the expression of which is dependent on crp gene function.

Amino Acid Sequence↗

PubMed1973

Chromosome replication during meiosis: identification of gene functions required for premeiotic DNA synthesis.

Recent comparisons of chromosome replication in meiotic and mitotic cells have revealed significant differences in both the rate and pattern of DNA synthesis during the final duplication preceding meiosis. These differences suggested that unique gene functions might be required for premeiotic replication that were not necessary for replication during growth. To provide evidence for such functions, we isolated stage-specific mutants in the yeast Saccharomyces cerevisiae which permitted vegetative replication but blocked the round of replication before meiosis. The mutants synthesized carbohydrate, protein, and RNA during the expected interval of premeiotic replication, suggesting that their lesions preferentially affected synthesis of DNA. The mutations blocked meiosis, as judged by a coincident inhibition of intragenic recombination and ascospore formation. The lesions were characterized as recessive nuclear genes, and were designated mei-1, mei-2, and mei-3; complementation indicated that the relevant gene products were not identical.

Aneuploidy↗

PubMed1999

Nucleic acid synthesis for investigations of gene functions.

The chemical syntheses of genes encoding human c-Ha-ras and T4 endonuclease V are described. These genes have been used for studies of mutagenesis by damaged bases and recognition of nucleic acids by proteins. The modes of interaction of photo-damaged DNA and cognate antibodies have also been investigated by biochemical and physicochemical approaches.

DNA Damage↗

PubMed1978

Simian virus 40 A gene function: DNA content analysis of Chinese hamster cells transformed by an early temperature-sensitive virus mutant.

Replication of two Chinese hamster embryo cell lines transformed by an early temperature-sensitive mutant of simian virus 40, tsA58, was examined by flow microfluorometry and autoradiography of [3H]thymidine-labeled cells in order to determine whether transformed cell DNA synthesis is initiated by the virus A gene. At the permissive temperature (37 degrees), cells transformed by the mutant were like the wild-type virus transformants in appearance, colony-forming ability, high saturation density, and rapid replication. At the nonpermissive temperature (40.5 degrees), the tsA58 transformed cells resembled normal embryo fibroblasts and seem to return to normal growth patterns. Although both mutant transformed cell lines at 40.5 degrees appeared to cease growth at low saturation density, the cells did not enter a resting state, but continued to replicate. The cultures were maintained at low densities by a balance among cell replication, cell death, and sloughing of dead cells into the supernatant. These results suggest that the simian virus 40 A gene function effected by the tsA58 mutation does not prevent Chinese hamster embryo transformed cells from entering a resting state, although the gene may control other phenotypic characteristics of transformation.

Autoradiography↗

PubMed1981

Parallel cross-reactivity patterns of 2 sets of antigenically distinct cytochrome c peptides: possible evidence for a presentational model of Ir gene function.

B10.A mice were immunized with either the carboxyl terminal peptide fragment 81-104 of pigeon cytochrome c or its acetimidyl derivative and an immune response was seen with strong preference for the immunogen. Strain distribution studies and blocking with an anti-Ia monoclonal antibody indicated that the same immune response (Ir) gene and restriction element were utilized in both responses. The specificity of the responses were evaluated by restimulating in vitro with a set of cytochrome c fragments from various species. Even though the derivatized and native fragments were poorly cross-reactive, the same phylogenetic pattern was seen when pigeon cytochrome c fragment 81-104 primed cells were tested with the set of underivatized fragments and when acetimidyl pigeon cytochrome c fragment 81-104 primed cells were tested with the same set of derivatized fragments. Primed cells from a 2nd major histocompatibility complex congenic strain of mice, B10.A(5R), displayed equivalent discrimination between derivatized and native forms but showed a markedly different phylogenetic pattern of cross-reactivity. These data indicate that the immune system recognizes 2 sites on the nominal antigen. One site, which accounts for the common hierarchy and is under Ir gene control, contains residues Gln-100, and possibly other carboxyl terminal residues. The 2nd site, which effects the distinction between native and derivatized fragments, contains at least 1 lysine other than at the carboxyl terminal. The implications of these data for theories of T cell recognition and Ir gene function are discussed.

Animals↗

PubMed1986

Regulation of human interleukin-2 gene: functional DNA sequences in the 5' flanking region for the gene expression in activated T lymphocytes.

Interleukin-2 (IL-2) is a lymphokine that plays a crucial role in the immune system, especially in the growth control of T lymphocytes. Expression of this lymphokine is restricted to activated T lymphocytes. Here we demonstrate the presence of unique DNA sequences in the 5' flanking region of the human IL-2 gene that control induced T-cell-specific gene expression. We also show that the DNA sequences function in an orientation-independent manner and activate a heterologous promoter which is otherwise inert in induced T cells. The DNA, which spans about 200 bp, contains regions with sequence homology to LTR sequences of HTLV-III (or LAV) and the 5' upstream region of the IL-2 receptor and interferon-gamma genes.

Base Sequence↗