Showing papers on "Gene published in 1989"

Dynamics of mitochondrial DNA evolution in animals: Amplification and sequencing with conserved primers (cytochrome b/12S ribosomal DNA/control region/evolutionary genetics/molecular phylogenies)

Abstract: With a standard set of primers directed toward conserved regions, we have used the polymerase chain reaction to amplify homologous segments ofmtDNA from more than 100 animal species, including mammals, birds, amphib- ians, fishes, and some invertebrates. Amplification and direct sequencing were possible using unpurified mtDNA from nano- gram samples of fresh specimens and microgram amounts of tissues preserved for months in alcohol or decades in the dry state. The bird and fish sequences evolve with the same strong bias toward transitions that holds for mammals. However, because the light strand of birds is deficient in thymine, thymine to cytosine transitions are less common than in other taxa. Amino acid replacement in a segment of the cytochrome b gene is faster in mammals and birds than in fishes and the pattern of replacements fits the structural hypothesis for cytochrome b. The unexpectedly wide taxonomic utility ofthese primers offers opportunities for phylogenetic and population research.

Mutations in the p53 gene occur in diverse human tumour types

Abstract: The p53 gene has been a constant source of fascination since its discovery nearly a decade ago. Originally considered to be an oncogene, several convergent lines of research have indicated that the wild-type gene product actually functions as a tumour suppressor gene. For example, expression of the neoplastic phenotype is inhibited, rather than promoted, when rat cells are transfected with the murine wild-type p53 gene together with mutant p53 genes and/or other oncogenes. Moreover, in human tumours, the short arm of chromosome 17 is often deleted. In colorectal cancers, the smallest common region of deletion is centred at 17p13.1; this region harbours the p53 gene, and in two tumours examined in detail, the remaining (non-deleted) p53 alleles were found to contain mutations. This result was provocative because allelic deletion coupled with mutation of the remaining allele is a theoretical hallmark of tumour-suppressor genes. In the present report, we have attempted to determine the generality of this observation; that is, whether tumours with allelic deletions of chromosome 17p contain mutant p53 genes in the allele that is retained. Our results suggest that (1) most tumours with such allelic deletions contain p53 point mutations resulting in amino-acid substitutions, (2) such mutations are not confined to tumours with allelic deletion, but also occur in at least some tumours that have retained both parental 17p alleles, and (3) p53 gene mutations are clustered in four 'hot-spots' which exactly coincide with the four most highly conserved regions of the gene. These results suggest that p53 mutations play a role in the development of many common human malignancies.

Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA

Abstract: Using a set of synthetic oligonucleotides homologous to broadly conserved sequences in-vitro amplification via the polymerase chain reaction followed by direct sequencing results in almost complete nucleotide determination of a gene coding for 16S ribosomal RNA. As a model system the nucleotide sequence of the 16S rRNA gene of M.kansasii was determined and found to be 98.7% homologous to that of M.bovis BCG. This is the first report on a contiguous sequence information of an entire amplified gene spanning 1.5 kb without any subcloning procedures.

Altering the genome by homologous recombination.

Abstract: Homologous recombination between DNA sequences residing in the chromosome and newly introduced, cloned DNA sequences (gene targeting) allows the transfer of any modification of the cloned gene into the genome of a living cell. This article discusses the current status of gene targeting with particular emphasis on germ line modification of the mouse genome, and describes the different methods so far employed to identify those rare embryonic stem cells in which the desired targeting event has occurred.

Chromosome 17 deletions and p53 gene mutations in colorectal carcinomas

Abstract: Previous studies have demonstrated that allelic deletions of the short arm of chromosome 17 occur in over 75% of colorectal carcinomas. Twenty chromosome 17p markers were used to localize the common region of deletion in these tumors to a region contained within bands 17p12 to 17p13.3. This region contains the gene for the transformation-associated protein p53. Southern and Northern blot hybridization experiments provided no evidence for gross alterations of the p53 gene or surrounding sequences. As a more rigorous test of the possibility that p53 was a target of the deletions, the p53 coding regions from two tumors were analyzed; these two tumors, like most colorectal carcinomas, had allelic deletions of chromosome 17p and expressed considerable amounts of p53 messenger RNA from the remaining allele. The remaining p53 allele was mutated in both tumors, with an alanine substituted for valine at codon 143 of one tumor and a histidine substituted for arginine at codon 175 of the second tumor. Both mutations occurred in a highly conserved region of the p53 gene that was previously found to be mutated in murine p53 oncogenes. The data suggest that p53 gene mutations may be involved in colorectal neoplasia, perhaps through inactivation of a tumor suppressor function of the wild-type p53 gene.

The E6 and E7 genes of the human papillomavirus type 16 together are necessary and sufficient for transformation of primary human keratinocytes.

Abstract: The early human papillomavirus type 16 genes that directly participate in the in vitro transformation of primary human keratinocytes have been defined. In the context of the full viral genome, mutations in either the E6 or E7 open reading frame completely abrogated transformation of these cells. Mutations in the E1, E2, and E2-E4 open reading frames, on the other hand, had no effect. Thus, both the full-length E6 and E7 genes were required for the induction of keratinocyte immortalization and resistance to terminal differentiation. The E6 and E7 genes expressed together from the human beta-actin promoter were sufficient for this transformation; mutation of either gene in the context of this recombinant plasmid eliminated the ability to induce stable differentiation-resistant transformants.

The HIV-1 rev trans-activator acts through a structured target sequence to activate nuclear export of unspliced viral mRNA.

Abstract: HUMAN immunodeficiency virus type 1 (HIV-1) replication requires the expression of two classes of viral mRNA. The early class of HIV-1 transcripts is fully spliced and encodes viral regulatory gene products. The functional expression of one of these nuclear regulatory proteins, termed Rev (formerly Art or Trs), induces the cytoplasmic expression of the incompletely spliced, late class of HIV-1 mRNAs that encode the viral structural proteins, including Gag and Env1–6. Here, we provide evidence that this induction reflects the export from the cell nucleus to the cytoplasm of a pool of unspliced viral RNA constitutively expressed in the nucleus. The hypothesis that Rev acts on RNA transport, rather than splicing, is further supported by the observation that the cytoplasmic expression of a non-spliceable HIV-1 env gene sequence is also subject to Rev regulation. Here we show that this Rev response requires a specific target sequence which coincides with a complex RNA secondary structure present in the env gene. The response to Rev is fully maintained when this sequence is relocated to other exonic or intronic locations within env but is ablated by inversion. These results indicate that the HIV-1 rev gene product induces HIV-1 structural gene expression by activating the sequence-specific nuclear export of incompletely spliced HIV-1 RNA species.

RFLP Mapping in Plant Breeding: New Tools for an Old Science

Abstract: Breeders have traditionally improved plant varieties by selecting on the basis of phenotype. Now restriction fragment length polymorphism (RFLP) linkage maps are being constructed for most major crop plants and these maps provide a more direct method for selecting desirable genes via their linkage to easily detectable RFLP markers. The integration of RFLP techniques into plant breeding promises to: (1) Expedite the movement of desirable genes among varieties, (2) Allow the transfer of novel genes from related wild species, (3) Make possible the analysis of complex polygenic characters as ensembles of single Mendelian factors, and (4) Establish genetic relationships between sexually incompatible crop plants. In the future, high density RFLP maps may also make it possible to clone genes whose products are unknown, such as genes for disease resistance or stress tolerance.

p53: a frequent target for genetic abnormalities in lung cancer

Abstract: Allele loss is a hallmark of chromosome regions harboring recessive oncogenes. Lung cancer frequently demonstrates loss of heterozygosity on 17p. Recent evidence suggests that the p53 gene located on 17p13 has many features of such an antioncogene. The p53 gene was frequently mutated or inactivated in all types of human lung cancer. The genetic abnormalities of p53 include gross changes such as homozygous deletions and abnormally sized messenger RNAs along with a variety of point or small mutations, which map to the p53 open reading frame and change amino acid sequence in a region highly conserved between mouse and man. In addition, very low or absent expression of p53 messenger RNA in lung cancer cell lines compared to normal lung was seen. These findings, coupled with the previous demonstration of 17p allele loss in lung cancer, strongly implicate p53 as an anti-oncogene whose disruption is involved in the pathogenesis of human lung cancer.

Identification of four conserved motifs among the RNA-dependent polymerase encoding elements.

Abstract: Four consensus sequences are conserved with the same linear arrangement in RNA-dependent DNA polymerases encoded by retroid elements and in RNA-dependent RNA polymerases encoded by plus-, minus- and double-strand RNA viruses. One of these motifs corresponds to the YGDD span previously described by Kamer and Argos (1984). These consensus sequences altogether lead to 4 strictly and 18 conservatively maintained amino acids embedded in a large domain of 120 to 210 amino acids. As judged from secondary structure predictions, each of the 4 motifs, which may cooperate to form a well-ordered domain, places one invariant amino acid in or proximal to turn structures that may be crucial for their correct positioning in a catalytic process. We suggest that this domain may constitute a prerequisite 'polymerase module' implicated in template seating and polymerase activity. At the evolutionary level, the sequence similarities, gap distribution and distances between each motif strongly suggest that the ancestral polymerase module was encoded by an individual genetic element which was most closely related to the plus-strand RNA viruses and the non-viral retroposons. This polymerase module gene may have subsequently propagated in the viral kingdom by distinct gene set recombination events leading to the wide viral variety observed today.

Lutropin-choriogonadotropin receptor: an unusual member of the G protein-coupled receptor family.

Abstract: A complementary DNA (cDNA) for the rat luteal lutropin-choriogonadotropin receptor (LH-CG-R) was isolated with the use of a DNA probe generated in a polymerase chain reaction with oligonucleotide primers based on peptide sequences of purified receptor protein. As would be predicted from the cDNA sequence, the LH-CG-R consists of a 26-residue signal peptide, a 341-residue extracellular domain displaying an internal repeat structure characteristic of members of the leucine-rich glycoprotein (LRG) family, and a 333-residue region containing seven transmembrane segments. This membrane-spanning region displays sequence similarity with all members of the G protein-coupled receptor family. Hence, the LH-CG-R gene may have evolved by recombination of LRG and G protein-coupled receptor genes. Cells engineered to express LH-CG-R cDNA bind human choriogonadotropin with high affinity and show an increase in cyclic adenosine monophosphate when exposed to hormone. As revealed by RNA blot analysis and in situ hybridization, the 4.4-kilobase cognate messenger RNA is prominently localized in the rat ovary.

Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression.

Abstract: A method is described for the production of recombinant adeno-associated virus (AAV) stocks that contain no detectable wild-type helper AAV. The recombinant viruses contained only the terminal 191 nucleotides of the AAV chromosome bracketing a nonviral marker gene. trans-Acting AAV functions were provided by a helper DNA in which the terminal 191 nucleotides of the AAV chromosome were substituted with adenovirus terminal sequences. Although the helper DNA did not appear to replicate, it expressed AAV functions at a substantially higher level than did DNA molecules that contained neither AAV nor adenovirus termini. Since the recombinant viruses with AAV termini contained no sequence homology to the helper DNA, no wild-type AAV was generated by homologous recombination within infected cells. Since the terminal region of the AAV chromosome is required for replication and encapsidation, only recombinant DNAs were amplified and packaged into AAV virions. When human cells were infected at a high multiplicity with a recombinant virus carrying a drug resistance marker gene, approximately 70% of the infected cells gave rise to colonies stably expressing the marker. The recombinant virus gene was then used to generate drug-resistant human cell lines subsequent to infection. These cells contained stably integrated copies of the recombinant viral DNA which could be excised, replicated, and encapsidated by infection with wild-type AAV plus adenovirus. Thus, AAV gene expression is not required for normal integration of an infecting DNA containing AAV termini.

Nucleotide substitution at major histocompatibility complex class II loci: evidence for overdominant selection.

Abstract: To study the mechanism of maintenance of polymorphism at major histocompatibility complex (MHC) loci, synonymous and nonsynonymous (amino acid-altering) nucleotide substitutions in the putative antigen-recognition site (included in the first domain of the MHC molecule) and other regions of human and mouse class II genes were examined. In the putative antigen-recognition site, the rate of nonsynonymous substitution was found to exceed that of synonymous substitution, whereas in the second domain the former was significantly lower than the latter. In light of a previous theoretical study and parallel findings in class I MHC loci, we conclude that the unusually high degree of polymorphism at class II MHC loci is caused mainly by overdominant selection (heterozygote advantage) operating in the antigen-recognition site.

The structural and functional organization of the murine HOX gene family resembles that of Drosophila homeotic genes

Abstract: This paper reports the cloning of the fourth major murine homeogene complex, HOX-5. The partial characterization of this gene cluster revealed the presence of two novel genes (Hox-5.2, Hox-5.3) located at the 5' extremity of this complex. In situ hybridization experiments showed that these two genes are transcribed in very posterior domains during embryonic and foetal development. We also show that Hox-1.6, the gene located at the 3' most position in the HOX-1 complex, has a very anterior expression boundary during early development. These results clearly support the recently proposed hypothesis that the expression of murine Antp-like homeobox-containing genes along the antero-posterior developing body axis follows a positional hierarchy which reflects their respective physical positions within the HOX clusters, similar to that which is found for the Drosophila homeotic genes. Such a structural and functional organization is likely conserved in most vertebrates. Moreover, on the basis of sequence comparisons, we propose that the ordering of homeobox-containing genes within clusters has been conserved between Drosophila and the house mouse. Thus, very different body plans might be achieved, both in insects and vertebrates, by evolutionarily conserved gene networks possibly displaying similar regulatory interactions.

Direct and sensitive detection of a pathogenic protozoan, Toxoplasma gondii, by polymerase chain reaction.

Abstract: We applied the polymerase chain reaction to detection of the pathogenic protozoan Toxoplasma gondii based on our identification of a 35-fold-repetitive gene (the B1 gene) as a target. Using this procedure, we were able to amplify and detect the DNA of a single organism directly from a crude cell lysate. This level of sensitivity also allowed us to detect the B1 gene from purified DNA samples containing as few as 10 parasites in the presence of 100,000 human leukocytes. This is representative of the maximal cellular infiltration (10(5)/ml) in 1 ml of cerebrospinal fluid obtained from patients with toxoplasmic encephalitis. The B1 gene is present and conserved in all six T. gondii strains tested to date, including two isolates from patients with acquired immunodeficiency syndrome. No signal was detected by using this assay and DNAs from a variety of other organisms, including several which might be found in the central nervous system of an immunocompromised host. This combination of sensitivity and specificity should make detection of the B1 gene based on polymerase chain reaction amplification a very useful method for diagnosis of toxoplasmosis both in immunocompromised hosts and in congenitally infected fetuses.

Cloning of cDNA for the major DNA-binding protein of the erythroid lineage through expression in mammalian cells

Abstract: Genes expressed in erythroid cells contain binding sites for a cell-specific factor believed to be an important regulator for this haematopoietic lineage. Using high-level transient expression in mammalian cells, we have identified complementary DNA encoding the murine protein. The factor, a new member of the zinc-finger family of DNA-binding proteins, is restricted to erythroid cells at the level of RNA expression and is closely homologous between mouse and man.

Novel phytochrome sequences in Arabidopsis thaliana: structure, evolution, and differential expression of a plant regulatory photoreceptor family

Abstract: Phytochrome is a plant regulatory photoreceptor that mediates red light effects on a wide variety of physiological and molecular responses. DNA blot analysis indicates that the Arabidopsis thaliana genome contains four to five phytochrome-related gene sequences. We have isolated and sequenced cDNA clones corresponding to three of these genes and have deduced the amino acid sequence of the full-length polypeptide encoded in each case. One of these proteins (phyA) shows 65-80% amino acid sequence identity with the major, etiolated-tissue phytochrome apoproteins described previously in other plant species. The other two polypeptides (phyB and phyC) are unique in that they have low sequence identity (approximately 50%) with each other, with phyA, and with all previously described phytochromes. The phyA, phyB, and phyC proteins are of similar molecular mass, have related hydropathic profiles, and contain a conserved chromophore attachment region. However, the sequence comparison data indicate that the three phy genes diverged early in plant evolution, well before the divergence of the two major groups of angiosperms, the monocots and dicots. The steady-state level of the phyA transcript is high in dark-grown A. thaliana seedlings and is down-regulated by light. In contrast, the phyB and phyC transcripts are present at lower levels and are not strongly light-regulated. These findings indicate that the red/far light-responsive phytochrome photoreceptor system in A. thaliana, and perhaps in all higher plants, consists of a family of chromoproteins that are heterogeneous in structure and regulation.

Generation and selection of recombinant varied binding proteins

Abstract: In order to obtain a novel binding protein against a chosen target, DNA molecules, each encoding a protein comprising one of a family of similar potential binding domains and a structural signal calling for the display of the protein on the outer surface of a chosen bacterial cell, bacterial spore or phage (genetic package) are introduced into a genetic package. The protein is expressed and the potential binding domain is displayed on the outer surface of the package. The cells or viruses bearing the binding domains which recognize the target molecule are isolated and amplified. The successful binding domains are then characterized. One or more of these successful binding domains is used as a model for the design of a new family of potential binding domains, and the process is repeated until a novel binding domain having a desired affinity for the target molecule is obtained. In one embodiment, the first family of potential binding domains is related to bovine pancreatic trypsin inhibitor, the genetic package is M13 phage, and the protein includes the outer surface transport signal of the M13 gene III protein.

Evolutionary relationship of archaebacteria, eubacteria, and eukaryotes inferred from phylogenetic trees of duplicated genes

Abstract: All extant organisms are though to be classified into three primary kingdoms, eubacteria, eukaryotes, and archaebacteria. The molecular evolutionary studies on the origin and evolution of archaebacteria to date have been carried out by inferring a molecular phylogenetic tree of the primary kingdoms based on comparison of a single molecule from a variety of extant species. From such comparison, it was not possible to derive the exact evolutionary relationship among the primary kingdoms, because the root of the tree could not be determined uniquely. To overcome this difficulty, we compared a pair of duplicated genes, elongation factors Tu and G, and the alpha and beta subunits of ATPase, which are thought to have diverged by gene duplication before divergence of the primary kingdoms. Using each protein pair, we inferred a composite phylogenetic tree with two clusters corresponding to different proteins, from which the evolutionary relationship of the primary kingdoms is determined uniquely. The inferred composite trees reveal that archaebacteria are more closely related to eukaryotes than to eubacteria for all the cases. By bootstrap resamplings, this relationship is reproduced with probabilities of 0.96, 0.79, 1.0, and 1.0 for elongation factors Tu and G and for ATPase subunits alpha and beta, respectively. There are also several lines of evidence for the close sequence similarity between archaebacteria and eukaryotes. Thus we propose that this tree topology represents the general evolutionary relationship among the three primary kingdoms.

Avian-to-human transmission of the PB1 gene of influenza A viruses in the 1957 and 1968 pandemics.

Abstract: We determined the origin and evolutionary pathways of the PB1 genes of influenza A viruses responsible for the 1957 and 1968 human pandemics and obtained information on the variable or conserved region of the PB1 protein. The evolutionary tree constructed from nucleotide sequences suggested the following: (i) the PB1 gene of the 1957 human pandemic strain, A/Singapore/1/57 (H2N2), was probably introduced from avian species and was maintained in humans until 1968; (ii) in the 1968 pandemic strain, A/NT/60/68 (H3N2), the PB1 gene was not derived from the previously circulating virus in humans but probably from another avian virus; and (iii) a current human H3N2 virus inherited the PB1 gene from an A/NT/60/68-like virus. Nucleotide sequence analysis also showed that the avian PB1 gene was introduced into pigs. Hence, transmission of the PB1 gene from avian to mammalian species is a relatively frequent event. Comparative analysis of deduced amino acid sequences disclosed highly conserved regions in PB1 proteins, which may be key structures required for PB1 activities.

Expression of a large family of POU-domain regulatory genes in mammalian brain development.

Abstract: A novel region referred to as the POU-domain is present in two tissue-specific transcription factors, Pit-1 and Oct-2, that activate expression of genes specifying pituitary and lymphocyte phenotypes. We report the identification of multiple new members of a large family of POU-domain genes expressed in adult brain, and document that all the known mammalian POU-domain genes, including Pit-1 and Oct-2, are expressed widely in the developing nervous system.