Showing papers on "Genome published in 1987"

Structural and transcriptional analysis of human papillomavirus type 16 sequences in cervical carcinoma cell lines.

Abstract: We cloned and analyzed the integrated human papillomavirus type 16 (HPV-16) genomes that are present in the human cervical carcinoma cell lines SiHa and CaSki. The single HPV-16 genome in the SiHa line was cloned as a 10-kilobase (kb) HindIII fragment. Integration of the HPV-16 genome occurred at bases 3132 and 3384 with disruption of the E2 and E4 open reading frames (ORFs). An additional 52-base-pair deletion of HPV-16 sequences fused the E2 and E4 ORFs. the 5' portion of the disrupted E2 ORF terminated immediately in the contiguous human right-flanking sequences. Heteroduplex analysis of this cloned integrated viral genome with the prototype HPV-16 DNA revealed no other deletions, insertions, or rearrangements. DNA sequence analysis of the E1 ORF, however, revealed the presence of an additional guanine at nucleotide 1138, resulting in the fusion of the E1a and E1b ORFs into a single E1 ORF. Sequence analysis of the human flanking sequences revealed one-half of an Alu sequence at the left junction and a sequence highly homologous to the human O repeat in the right-flanking region. Analysis of the three most abundant BamHI clones from the CaSki line showed that these consisted of full-length, 7.9-kb HPV-16 DNA; a 6.5-kb genome resulting from a 1.4-kb deletion of the long control region; and a 10.5-kb clone generated by a 2.6-kb tandem repeat of the 3' early region. These HPV-16 genomes were arranged in the host chromosomes as head-to-tail, tandemly repeated arrays. Transcription analysis revealed expression of the HPV-16 genome in each of these two cervical carcinoma cell lines, albeit at significantly different levels. Preliminary mapping of the viral RNA with subgenomic strand-specific probes indicated that viral transcription appeared to be derived primarily from the E6 and E7 ORFs.

The guanine and cytosine content of genomic DNA and bacterial evolution

Abstract: The genomic guanine and cytosine (G + C) content of eubacteria is related to their phylogeny. The G + C content of various parts of the genome (protein genes, stable RNA genes, and spacers) reveals a positive linear correlation with the G + C content of their genomic DNA. However, the plotted correlation slopes differ among various parts of the genome or among the first, second, and third positions of the codons depending on their functional importance. Facts suggest that biased mutation pressure, called A X T/G X C pressure, has affected whole DNA during evolution so as to determine the genomic G + C content in a given bacterium. The role of A X T/G X C pressure in diversification of bacterial DNA sequences and codon usage patterns is discussed in the perspective of the neutral theory of molecular evolution.

DNA fingerprinting in birds.

Abstract: Several regions of the human genome are highly variable in populations because the number of repeats in these regions of a short 'minisatellite' sequence varies at high frequency. Different minisatellites have a core sequence1,2 in common, however, and probes made up of tandem repeats of this core sequence detect many highly variable DNA fragments in several species including humans1,3, cats4, dogs4 and mice5. The hypervariable sequences detected in this way are dispersed in the genome and their variability means that they can be used as a DNA 'fingerprint', providing a novel method for the identification of individuals2,6, confirmation of biological relationships7,8 and human genetic analysis9,10. We show here that human minisatellite-derived probes also detect highly variable regions in bird DNAs. Segregation analysis in a house sparrow family confirms that these regions comprise many mostly heterozygous dispersed loci and we conclude that house sparrow DNA fingerprints are analagous to those of humans. Fingerprint analysis identified one nestling, with fingerprint bands not present in the parent pair's fingerprints, which we conclude resulted from an extrapair copulation. Extrabond copulations have been described in many wild bird species11–13, but their success and hence adaptive significance have rarely been quantifiable14–20. DNA fingerprinting will be of great significance to studies of the sociobiology, demography and ecology of wild birds.

Genomic imprinting determines methylation of parental alleles in transgenic mice.

Abstract: Mouse embryogenesis relies on the presence of both the maternal and the paternal genome for development to term1,2. It has been proposed that specific modifications are imprinted onto the chromosomes during gametogenesis3; these modifications are stably propagated4, and their expression results in distinct and complementary contributions of the two parental genomes to the development of the embryo and the extraembryonic membranes5,6. Genetic data further suggest that a substantial proportion of the genome could be subject to chromosomal imprinting7, the molecular nature of which is unknown. We used random DNA insertions in transgenic mice to probe the genome for modified regions. The DNA methylation patterns of transgenic alleles were compared after transmission from mother or father in seven mouse strains carrying autosomal insertions of the same transgenic marker. One of these loci showed a clear difference in DNA methylation specific for its parental origin, with the paternally inherited copy being relatively undermethylated. This difference was observed in embryos on day 10 of gestation, but not in their extraembryonic membranes. Moreover, the methylation pattern was faithfully reversed upon each germline transmission to the opposite sex. Our findings provide evidence for heritable molecular differences between maternally and paternally derived alleles on mouse chromosomes.

Anti-sense regulation of gene expression in plant cells

Abstract: Regulation of expression of genes encoded for in plant cell genomes is achieved by integration of a gene under the transcriptional control of a promoter which is functional in the host and in which the trans­cribed strand of DNA is complementary to the strand of DNA that is transcribed from the endogenous gene(s) one wishes to regulate. The integrated gene, referred to as anti-sense, provides an RNA sequence capable of binding to naturally existing RNAs, exemplified by polygalacturonase, and inhibiting their expression, where the anti-sense sequence may bind to the coding, non-coding, or both, portions of the RNA. The anti-­sense construction may be introduced into the plant cells in a variety of ways and be integrated into the plant genome for inducible or constitutive transcrip­tion of the anti-sense sequence. A wide variety of plant cell properties may be modified by employing this technique.

A physical map of the Escherichia coli K12 genome.

Abstract: A physical map of a genome is the structure of its DNA. Construction of such a map is a first step in the complete characterization of that DNA. The restriction endonuclease Not I cuts the genome of Escherichia coli K12 into 22 DNA fragments ranging from 20 kilobases (20,000 base pairs) to 1000 kilobases. These can be separated by pulsed field gel electrophoresis. The order of the fragments in the genome was determined from available E. coli genetic information and analysis of partial digest patterns. The resulting ordered set of fragments is a macrorestriction map. This map facilitates genetic and molecular studies on E. coli, and its construction serves as a model for further endeavors on larger genomes.

Molecular analysis and chromosomal mapping of amplified genes isolated from a transformed mouse 3T3 cell line

Abstract: We are exploring the origin and function of amplified DNA sequences associated with double minutes (DMs) in a spontaneously transformed derivative of mouse 3T3 cells. Toward that goal, we have constructed a cDNA library using RNA from these cells and have isolated cDNA clones representing sequences that are amplified and overexpressed in these 3T3-DM cells. From results of Northern- and Southern-blot analyses, we conclude that these cDNAs represent two distinct genes, which we have designated mdm-1and mdm-2.Using DNAs from a panel of Chinese hamster-mouse somatic cell hybrids together with in situ hybridization protocols for gene mapping studies, we have found that these DM-associated, amplified DNA sequences originate from mouse chromosome 10, region C1–C3. Sequences homologous to mdm-1and mdm-2are present in the genomes of several species examined, including that of man.

Why do chloroplasts and mitochondria contain so many copies of their genome

Abstract: The very high genome copy number in cytoplasmic organelles is a puzzling fact in cell biology. It is proposed here that high copy number reflects an increased need for organellar ribosomes that can only be satisfied by the increased ribosomal RNA gene number that results from genome multiplication.

The mitochondrial genotype can influence nuclear gene expression in yeast

Abstract: Isochromosomal, respiratory-deficient yeast strains, such as a mit-, a hypersuppressive petite, and a petite lacking mitochondrial DNA, are phenotypically identical in spite of differences in their mitochondrial genomes. Subtractive hybridizations of complementary DNA's to polyadenylated RNA isolated from derepressed cultures of these strains reveal the presence of nuclear-encoded transcripts whose abundance varies not only between them and their respiratory-competent parent, but among the respiratory-deficient strains themselves. Transcripts of some nuclear-encoded mitochondrial proteins, like cytochrome c and the alpha and beta subunits of the mitochondrial adenosine triphosphatase, whose abundance is affected by glucose or heme, do not vary. In the absence of major metabolic variables, yeast cells seem to respond to the quality and quantity of mitochondrial DNA and modulate the levels of nuclear-encoded RNA's, perhaps as a means of intergenomic regulation.

Restriction endonucleases for pulsed field mapping of bacterial genomes

Abstract: Fundamental to many bacterial genome mapping strategies currently under development is the need to cleave the genome into a few large DNA fragments that can be resolved by pulsed field gel electrophoresis. Identification of endonucleases that infrequently cut a genome is of key importance in this process. We show that the tetranucleotide CTAG is extremely rare in most bacterial genomes with G+C contents above 45%. As a consequence, most of the sixteen bacterial genomes we have tested are cleaved less than once every 100,000 base pairs by one or more endonucleases that have CTAG in their recognition sequences: Xba I (TCTAGA), Spe I (ACTAGT), Avr II (CCTAGG) and Nhe I (GCTAGC). Similarly, CCG and CGG are the rarest trinucleotides in many genomes with G+C content of less than 45%. Thus, Sma I (CCCGGG), Rsr II (CGGWCCG), Nae I (GCCGGC) and Sac II (CCGCGG) are often suitable endonucleases for producing fragments that average over 100,000 base pairs from such genomes. Pulsed field gel electrophoresis of the fragments that result from cleavage with endonucleases that cleave only a few times per genome should assist in the physical mapping of many prokaryotic genomes.

The Mitochondrial Genome of Kinetoplastid Protozoa: Genomic Organization, Transcription, Replication, and Evolution

Abstract: INTRODUCTION 363 KI.NETOPLAST MINICIRCLE DNA 365 Is There a Gene Product? 365 Sequence Organization in Different Species 365 Sequence Heterogeneity as a Diagnostic and Epidemiological Tool 368 Analysis of Conformational Bend in Minicircle DNA 370 Replication of Minicircle DNA 371 KINETOPLAST .MAXICIRCLE DNA 372 Mitochondrial Ribosomal R.VA genes 372 Mitochondrial Siruciural Genes and Open Reading Frames 372 Comparison of the Maxicircle Genomes from Two Kinetoplastid Species 373 The c o n Frameshift 374 Divergence of .Maxicircle Genes from Mitochondrial Genes of Other Organisms 374 The Divergent Region cf the .Maxicircle 374 Transcription (^Maxicircle Genes 375 Regulation of Maxicircle Transcription During the Life Cycle of Trypanosoma bnicei 375 CONCLUSIONS 377

Bizarre tRNAs inferred from DNA sequences of mitochondrial genomes of nematode worms.

Abstract: The complete nucleotide sequence of the mitochondrial DNA (mtDNA) molecule of the parasitic nematode worm Ascaris suum has been determined. This molecule lacks genes for tRNAs of the standard form. Instead, 21 sequences are found that can be folded into structures that resemble tRNAs in which the T psi C arm and variable loop are missing and replaced with a single loop of between 4 and 12 nucleotides. Considerations of various properties of these sequences, including the number, predicted anticodons, conserved nucleotides, direction of transcription, base composition, and relative gene arrangements are consistent with the interpretation that they are genes for a different sort of tRNA. Transfer RNA genes with a similar potential secondary structure are found in mtDNA of the free-living nematode Caenorhabditis elegans, suggesting that this unusual form of tRNA is used by all nematode mitochondria.

Genomic organization of alpha satellite DNA on human chromosome 7: evidence for two distinct alphoid domains on a single chromosome.

Abstract: A complete understanding of chromosomal disjunction during mitosis and meiosis in complex genomes such as the human genome awaits detailed characterization of both the molecular structure and genetic behavior of the centromeric regions of chromosomes. Such analyses in turn require knowledge of the organization and nature of DNA sequences associated with centromeres. The most prominent class of centromeric DNA sequences in the human genome is the alpha satellite family of tandemly repeated DNA, which is organized as distinct chromosomal subsets. Each subset is characterized by a particular multimeric higher-order repeat unit consisting of tandemly reiterated, diverged alpha satellite monomers of approximately 171 base pairs. The higher-order repeat units are themselves tandemly reiterated and represent the most recently amplified or fixed alphoid sequences. We present evidence that there are at least two independent domains of alpha satellite DNA on chromosome 7, each characterized by their own distinct higher-order repeat structure. We determined the complete nucleotide sequences of a 6-monomer higher-order repeat unit, which is present in approximately 500 copies per chromosome 7, as well as those of a less-abundant (approximately 10 copies) 16-monomer higher-order repeat unit. Sequence analysis indicated that these repeats are evolutionarily distinct. Genomic hybridization experiments established that each is maintained in relatively homogeneous tandem arrays with no detectable interspersion. We propose mechanisms by which multiple unrelated higher-order repeat domains may be formed and maintained within a single chromosomal subset.

Novel transcription map for the B19 (human) pathogenic parvovirus.

Abstract: The B19 parvovirus, a small single-stranded DNA virus of 5.4 kilobases, is pathogenic in humans. B19 has remarkable specificity for erythroid progenitor cells and has been propagated in vitro only with human erythroid bone marrow. Replication of viral DNA and the viral protein products of B19 appear similar to those of other animal parvoviruses. However, B19 transcription had unusual features in comparison with that in other animal parvoviruses. At least nine overlapping poly(A)+ transcripts were identified in infected cells; all but one contained large introns. B19 differed from other parvoviruses in the initiation of all transcripts at a strong left side promoter (p6) and the absence of a functional internal promoter; the presence of short 5' leader sequences of about 60 bases and very large introns for RNAs encoded by the right side of the genome; two separate transcription termination sites, in contrast to cotermination at the far right side of the genome for other parvoviruses; the probable utilization by three transcripts of a variant polyadenylation signal (ATTAAA or AATAAC) in the middle of the genome; and the abundance of two unique transcripts from the middle of the genome which did not code for capsid proteins. The unusual transcription map of B19 suggests that regulation of the relative abundance of transcripts occurs by splicing and termination-polyadenylation events rather than by promoter strength. In combination with the published nucleotide sequence, the novel transcription map separated the pathogenic B19 virus at a molecular level from other animal parvoviruses and human adeno-associated virus.

Developmental correlates of genome size in plethodontid salamanders and their implications for genome evolution.

Abstract: We present an analysis of the evolutionary relationship between genome size (C-value, mass of DNA per haploid nucleus) and developmental rate using observations of limb regeneration in salamanders of the family Plethodontidae. Rates of growth and differentiation of regenerating limbs are reported for 27 plethodontid species whose C-values range from 14 to 76 picograms. A phylogenetic analysis employing Felsenstein's method of independent contrasts indicates that rate of differentiation is inversely proportional to genome size, although we have not identified any statistically significant association between genome size and the growth rate of regenerating tissue. Our results are consistent with an interpretation that genome size may place a limit on the maximum rate of regeneration attainable in plethodontid salamanders. The implications of our findings for the "junk DNA," "nucleotypic DNA," "selfish DNA," and "skeletal DNA" hypotheses of genome evolution are discussed.

Stimulation of expression of the human endogenous retrovirus genome by female steroid hormones in human breast cancer cell line T47D.

Abstract: Expression of the human endogenous retrovirus genome HERV-K, homologous to mouse mammary tumor virus, was investigated in cultured human tumor cells. In several cell lines, the HERV-K genome was expressed as an 8.8-kilobase poly(A)+ RNA which appeared to be a full-size transcript of this genome. In the human breast cancer cell line T47D, stimulation of HERV-K genome expression by progesterone was observed after estradiol treatment.

Small nuclear inclusion protein encoded by a plant potyvirus genome is a protease.

Abstract: Tobacco etch virus, a plant potyvirus, expresses its RNA genome as a large polyprotein precursor which undergoes extensive proteolytic processing to yield seven or more mature products Two of these products, proteins with apparent molecular weights of 49,000 and 54,000 (49K and 54K proteins), aggregate in the form of crystalline inclusions within the nuclei of infected cells Cell-free translation of synthetic transcripts was used to map the genes for these two products on the viral genome and to express an enzymatically active protein The 49K protein was determined to be a viral protease responsible for several cleavages of the polyprotein, including its own autocatalytic excision Analyses of products expressed from the 49K protein genes which were altered by deletion revealed that only the carboxyl-terminal half was required for proteolytic activity

RNA from an immediate early region of the type 1 herpes simplex virus genome is present in the trigeminal ganglia of latently infected mice.

Abstract: Transcription of the type 1 herpes simplex virus (HSV-1) genome in trigeminal ganglia of latently infected mice was studied using in situ hybridization. Probes representative of each temporal gene class were used to determine the regions of the genome that encode the transcripts present in latently infected cells. Probes encoding HSV-1 sequences of the five immediate early genes and representative early (thymidine kinase), early-late (major capsid protein), and late (glycoprotein C) genes were used in these experiments. Of the probes tested, only those encoding the immediate early gene product infected-cell polypeptide (ICP) 0 hybridized to RNA in latently infected tissues. Probes containing the other immediate early genes (ICP4, ICP22, ICP27, and ICP47) and the representative early, early-late, and late genes did not hybridize. Two probes covering approximately equal to 30% of the HSV-1 genome and encoding over 20 early and late transcripts also did not hybridize to RNA in latently infected tissues. These results, with probes spanning greater than 60% of the HSV-1 genome, suggest that transcription of the HSV-1 genome is restricted to one region in latently infected mouse trigeminal ganglia.

Genome similarities between plant and animal RNA viruses.

Abstract: Gene sequence comparisons and protein comparisons provide more and more evidence that evolutionary links exist between plant and animal RNA viruses, irrespective of whether they have an isometric or rod-shaped particle, or of whether they have a divided or non-divided genome. Although a phylogeny based on these molecular data cannot yet be constructed the results obtained so far shed a new light on the origin and evolution of RNA viruses and are important in terms of their taxonomy.

Chloroplast DNA from lettuce and Barnadesia (Asteraceae): structure, gene localization, and characterization of a large inversion

Abstract: We have cloned into plasmids 17 of 18 lettuce chloroplast DNA SacI fragments covering 96% of the genome. The cloned fragments were used to construct cleavage maps for 10 restriction enzymes for the chloroplast genomes of lettuce (Lactuca sativa) and Barnadesia caryophylla, two distantly related species in the sunflower family (Asteraceae). Both genomes are approximately 151 kb in size and contain a 25 kb inverted repeat. We also mapped the position and orientation of 37 chloroplast DNA genes. The mapping studies reveal that chloroplast DNAs of lettuce and Barnadesia differ by a 22 kb inversion in the large single copy region. Barnadesia has retained the primitive land plant genome arrangement, while the inversion has occurred in a lettuce lineage. The endpoints of the derived lettuce inversion were located by comparison to the well-characterized spinach and tobacco genomes. Both endpoints are located in intergenic spacers within tRNA gene clusters; one cluster being located downstream from the atpA gene and the other upstream from the psbD gene. The endpoint near the atpA gene is very close to one endpoint of a 20 kb inversion in wheat (Howe et al. 1983; Quigley and Weil 1985). Comparison of the restriction site maps gives an estimated sequence divergence of 3.7% for the lettuce and Barnadesia genomes. This value is relatively low compared to previous estimates for other angiosperm groups, suggesting a high degree of sequence conservation in the Asteraceae.

The mode of hepatitis B virus DNA integration in chromosomes of human hepatocellular carcinoma.

Abstract: Nineteen DNA samples that carry integrated hepatitis B virus (HBV) DNA were isolated from seven independent human hepatomas by molecular cloning, and their structures were determined. The results, combined with reported data, were analyzed so that one can obtain insights into the mechanisms of integration of this virus DNA and possible rearrangements that occur subsequently. The distribution of DNA junctions along the virus genome suggests that there are recombination-proficient regions. Thus, about half of the integrants were the Coh type, viz., one of their virus-cell DNA junctions fell within the so-called cohesive end region that lies between two 11-bp direct repeats (DR1 and DR2) in the virus genome where transcription and replication of the genome are initiated. All the integrated virus genomes were defective at least in one site around the cohesive end region, particularly within the X gene. The recombination-proficient regions are used not only for formation of virus-cell but also of virus-virus junctions. Neither virus nor cell DNA show unique sequences at the junctions, and targets for integration lie on many different chromosomes.

SSC1, a member of the 70-kDa heat shock protein multigene family of Saccharomyces cerevisiae, is essential for growth

Abstract: The genome of the yeast Saccharomyces cerevisiae contains a family of genes related to the HSP70 genes (encoding the 70-kDa heat shock protein) of other eukaryotes. Mutations in two of these yeast genes (SSC1 and SSD1), whose expression is increased a few fold after temperature upshift, were constructed in vitro and substituted into the yeast genome in place of the wild-type alleles. No phenotypic effects of the mutation in SSD1 were detected. However, a functional SSC1 gene is essential for vegetative growth. This result, in conjunction with experiments involving mutations in other members of this multigene family, indicates that at least three distinct functions are carried out by genes of the HSP70 family.

Chloroplast DNA evolution among legumes: Loss of a large inverted repeat occurred prior to other sequence rearrangements

Abstract: We have compared the sequence organization of four previously uncharacterized legume chloroplast DNAs - from alfalfa, lupine, wisteria and subclover — to that of legume chloroplast DNAs that either retain a large, ribosomal RNA-encoding inverted repeat (mung bean) or have deleted one half of this repeat (broad bean). The circular, 126 kilobase pair (kb) alfalfa chloroplast genome, like those of broad bean and pea, lacks any detectable repeated sequences and contains only a single set of ribosomal RNA genes. However, in contrast to broad bean and pea, alfalfa chloroplast DNA is unrearranged (except for the deletion of one segment of the inverted repeat) relative to chloroplast DNA from mung bean. Together with other findings reported here, these results allow us to determine which of the four possible inverted repeat configurations was deleted in the alfalfa-pea-broad bean lineage, and to show how the present-day broad bean genome may have been derived from an alfalfa-like ancestral genome by two major sequence inversions. The 147 kb lupine chloroplast genome contains a 22 kb inverted repeat and has essentially complete colinearity with the mung bean genome. In contrast, the 130 kb wisteria genome has deleted one half of the inverted repeat and appears colinear with the alfalfa genome. The 140 kb subclover genome has been extensively rearranged and contains a family of at least five dispersed repetitive sequence elements, each several hundred by in size; this is the first report of dispersed repeats of this size in a land plant chloroplast genome. We conclude that the inverted repeat has been lost only once among legumes and that this loss occurred prior to all the other rearrangements observed in subclover, broad bean and pea. Of those lineages that lack the inverted repeat, some are stable and unrearranged, other have undergone a moderate amount of rearrangement, while still others have sustained a complex series of rearrangement either with or without major sequence duplications and transpositions.

Clustering of genes dispensable for growth in culture in the S component of the HSV-1 genome

Abstract: The herpes simplex virus 1 genome consists of one long and one short stretch of unique sequences flanked by inverted repeat sequences The nucleotide sequence and RNA map predict 12 open reading frames designated as US1 through US12 within the short stretch of unique sequences This paper reports the construction of virus mutants from which US2, US3, or US4 had been deleted that are capable of growth in cell culture One of the three deleted genes, US4, specifies the viral envelope glycoprotein G Mutants with deletions in US1, US8, US9, US10, US11, and US12 have been previously reported The nine genes deleted from this region form two clusters, US1 through US4 and US8 through US12, and encode at least two and possibly more structural proteins The presence of so many genes dispensable for growth in cell culture suggests several hypotheses regarding their function and evolution