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PubMed2004

A reciprocal translocation, induced by a canonical integration of a single T-DNA, interrupts the HMG-I/Y Arabidopsis thaliana gene.

Major chromosomal rearrangements occur during Arabidopsis thaliana T-DNA transformation. They generally result from interactions between multiple T-DNA copies during the integration process or from aborted integration events. We report here a reciprocal translocation associated with the integration of a single T-DNA which otherwise shows all the characteristic features of a canonical integration event. The exchanged fragments roughly correspond to half of the left arm of chromosome 1 and to two thirds of the right arm of chromosome 2. The chromosome 1 breakpoint maps close to position 23.6 cM and interrupts the coding sequence of the HMG-I/Y gene, which is present at a single copy in the Arabidopsis genome and encodes a non-histone chromosomal protein putatively involved in regulation of gene expression. The chromosome 2 breakpoint maps close to position 33.6 cM, and is located 419 bp upstream of a gene encoding a putative homeodomain transcription factor. Homozygotes for the translocation display a severe phenotype with major developmental abnormalities and total sterility, while heterozygotes are fertile, most of them showing a wild-type phenotype. Among the six possible unbalanced genotypic classes, four are entirely lethal while only a few individuals from the two others survive. Analysis of relations between phenotypes and genotypes strongly suggests that the major phenotypic alterations observed do not result from inactivation of the HMG-I/Y gene.

Arabidopsis↗
PubMed1995

Spliced HERV-H endogenous retroviral sequences in human genomic DNA: evidence for amplification via retrotransposition.

HERV-H elements are a large family of endogenous retrovirus-like sequences found in approximately 1000 dispersed copies in the genomes of humans and other primates. The most abundant subclass of these elements is a partially deleted form of 5.8 kb which is transcribed primarily as a 5.6-kb unit length RNA and a 3.7-kb spliced derivative. The provirus-like structure of these elements suggests that their numbers have increased in the genome through retrotransposition. However, this has not been demonstrated for HERV-H. To determine if genomic expansion of HERV-H elements involved an RNA intermediate, primate DNAs were screened by PCR for elements that were transcribed, spliced, reverse transcribed, and integrated back into the genome. This PCR screen detected several genomic HERV-H fragments that appear to be derived from spliced transcripts. Interestingly, the presence of one of these fragments is polymorphic in humans, suggesting that its integration was a relatively recent event. Another PCR strategy was used to determine that at least one of the spliced elements has an intact 5' LTR, indicating that it is not simply a "processed pseudogene" or cDNA copy of a HERV-H transcript. Genomic cloning and sequencing of a human locus harboring a spliced element revealed the expected structure, e.g., intact LTRs and flanking 5-bp direct repeats, for a virally retrotransposed element. A genomic library screening method also indicated that very few HERV-H elements (less than 1%) have the structure of processed pseudogenes. These results suggest that most HERV-H elements amplified in the genome as viral retrotransposons.

Animals↗
PubMed2003

Drift as a mechanism for cultural change: an example from baby names.

In the social sciences, there is currently no consensus on the mechanism by which cultural elements come and go in human society. For elements that are value-neutral, an appropriate null model may be one of random copying between individuals in the population. We show that the frequency distributions of baby names used in the United States in each decade of the twentieth century, for both males and females, obey a power law that is maintained over 100 years even though the population is growing, names are being introduced and lost every decade and large changes in the frequencies of specific names are common. We show that these distributions are satisfactorily explained by a simple process in which individuals randomly copy names from each other, a process that is analogous to the infinite-allele model of population genetics with random genetic drift. By its simplicity, this model provides a powerful null hypothesis for cultural change. It further explains why a few elements inevitably become highly popular, even if they have no intrinsic superiority over alternatives. Random copying could potentially explain power law distributions in other cultural realms, including the links on the World Wide Web.

Culture↗
PubMed1989

Synthesis of a homodimer neurohormone precursor of locust adipokinetic hormone studied by in vitro translation and cDNA cloning.

The homodimer neurohormone precursor P1, consisting of 41 residue subunits or A-chains, is synthesized by the glandular neurosecretory cells of the corpora cardiaca (CC) of the locust Schistocerca gregaria. Processing of P1 generates two copies of a 10 amino acid peptide neurohormone (AKH I) and one copy of a homodimer peptide (APRP 1). Here we show that the P1 dimer is formed from two independent A-chain translation products. Translation of CC mRNA in vitro produces a prominent 6.4 kd protein, the synthesis of which can be blocked by oligonucleotides hybridizing to mRNA encoding the A-chain. Northern blot experiments suggest that the 6.4 kd protein is produced by an integral of 500 base mRNA. cDNA cloning reveals a pre-A-chain structure in which a single copy of the A-chain is preceded by a 22 amino acid signal peptide. This evidence indicates that the P1 dimer is synthesized by coupling of very small translational products rather than by folding and processing of a larger protein containing more than one copy of the A-chain.

Amino Acid Sequence↗
PubMed1998

Song learning in brood-parasitic indigobirds Vidua chalybeata: song mimicry of the host species.

Brood-parasitic village indigobirds, Vidua chalybeata, were bred in captivity and foster-reared by their normal host, red-billed firefinch, Lagonosticta senegala, or by an experimental foster species, Bengalese finch, Lonchura striata. Male indigobirds reared by Bengalese finches developed the songs of Bengalese finches, and males reared by firefinches developed songs of firefinches. Males copied their foster father only when they had lived with him long after independence (45 days post-fledging), while males separated normally at independence (22-24 days post-fledging) copied songs of other individuals and not songs of their foster father. Males reared by Bengalese finches showed no preference to learn firefinch song over songs of the experimental foster species or other control finch species even when they had lived with firefinches as companions from the time of fledging to independence. Males copied several song themes, acquired the same number of mimicry songs, and acquired their songs at the same age, whether reared by Bengalese finches or by firefinches. When they lived with other indigobirds, the male indigobirds copied mimicry songs of male indigobirds that mimicked the same foster species. We predicted mimicry-song specificity and repertoire size in experimental indigobirds from a hypothesis of an early developmental period when young indigobirds focus their attention on their foster parents, and a later period when they direct their attention to other birds with similar songs. The predictions, based on field observations of wild birds, were that (1) males reared by a novel foster species other than the normal host would learn the song of that foster species, and (2) males that left their foster parents at the normal time of independence would copy the songs of other individuals, including other adult indigobirds that mimicked the same foster species. Begging calls of young indigobirds did not mimic the calls of young firefinches. Indigobirds reared alone, or with young of the normal host or of the experimental foster species, all developed begging calls in adult song that resembled their own begging as nestlings and fledglings, and only males that heard other adult indigobirds with firefinch-mimicry begging developed firefinch begging in their song. The incorporation of the innate begging calls as well as the learned begging calls into adult song, and the modification of the song themes of their individual song models, suggest that song development involves processes in addition to copying the songs of their own foster species and of older adult male indigobirds with songs like their own foster parents. Copyright 1998 The Association for the Study of Animal Behaviour. Copyright 1998 The Association for the Study of Animal Behaviour.

Journal Article↗
PubMed1983

Bypass of pyrimidine dimers in DNA of bacteriophage T4 via induction of primer RNA.

Bacteriophage T4 has a third pathway for repair of damaged DNA besides excision repair and recombination repair. This pathway is a mechanism for the toleration of lesions rather than the repair of lesions. The substrate for this process is gapped DNA copied from a damaged template. Evidence indicates that these gaps are filled, giving rise to daughter strands that are sensitive to heat and to treatments with RNAase. These daughter strands subsequently serve as templates for DNA that is resistant to RNAase. This third pathway is dependent upon gene 41 (RNA-priming protein), gene uvsZ (function unknown) and gene 30 (polynucleotide ligase) and is presumed to consist of 4 steps: (1) induction of primer RNA opposite the lesion in the template; (2) elongation of primers by DNA polymerase; (3) ligation of daughter-strand fragments, without removal of primer RNA; (4) replication of DNA carrying RNA sequences, giving homogeneous DNA strands. We have called this process 'Re-initiation repair'.

DNA Repair↗
PubMed1991

Macronuclear transformation with specific DNA fragments controls the content of the new macronuclear genome in Paramecium tetraurelia.

A previously isolated mutant cell line called d48 contains a complete copy of the A surface antigen gene in the micronuclear genome, but the gene is not incorporated into the macronucleus. Previous experiments have shown that a cytoplasmic factor made in the wild-type macronucleus can rescue the mutant. Recently, S. Koizumi and S. Kobayashi (Mol. Cell. Biol. 9:4398-4401, 1989) observed that injection of a plasmid containing the A gene into the d48 macronucleus rescued the cell line after autogamy. It is shown here that an 8.8-kb EcoRI fragment containing only a portion of the A gene coding region is sufficient for the rescue of d48. The inability of other A gene fragments to rescue the mutant shows that this effect is dependent upon specific Paramecium DNA sequences. Rescue results in restoration of the wild-type DNA restriction pattern in the macronucleus. These results are consistent with a model in which the macronuclear A locus normally makes an additional gene product that is required for correct processing of the micronuclear copy of the A gene.

Animals↗
PubMed1999

Development and evaluation of quantitative-competitive PCR for quantitation of coxsackievirus B3 RNA in experimentally infected murine tissues.

A method is described for quantitation of enterovirus RNA in experimentally infected murine tissues. Viral RNA was extracted from tissue samples and amplified by reverse transcriptase PCR in the presence of an internal standard RNA. The ratio of PCR product derived from viral RNA and internal standard RNA was then determined using specific probes in a post-PCR electrochemiluminescent hybridization assay. This provided an estimate of the viral RNA copy number in the original sample, and detection of PCR product derived from internal standard RNA validated sample processing and amplification procedures. RNA copy number correlated with viral infectivity of cell culture-derived virus, and one tissue culture infective dose was found to contain approximately 10(3) genome equivalents. The ratio of RNA copy number to infectivity in myocardial tissue taken from mice during the acute phase of coxsackievirus B3 myocarditis was more variable ranging from 10(4)-10(7), and was dependent on the stage of infection, reflecting differential rates of clearance for viral RNA and viral infectivity. The assay is rapid, and could facilitate investigations which currently rely upon enterovirus quantitation by titration in cell culture. This would be useful for experimental studies of viral pathogenesis, prophylaxis and antiviral therapy.

Acute Disease↗
PubMed1990

The structure of the gene for mouse filaggrin and a comparison of the repeating units.

Filaggrins are an important class of intermediate filament-associated proteins that are involved in the organization of keratin filaments in the terminal stages of mammalian epidermal differentiation. Filaggrins are initially synthesized as very large polyprotein precursors consisting of many tandemly arranged repeats that are later liberated by proteolytic processes to yield many copies of the functional protein. We have recently characterized a cDNA clone to mouse filaggrin (Rothnagel, J. A., Mehrel. T., Idler, W. W., Roop, D. R., and Steinert, P. M. (1987) J. Biol. Chem. 262, 15643-15648) which encodes a 750-base pair (250-amino acid) repeating element having properties consistent with a filaggrin molecule. Southern blot analysis of total mouse DNA and the mouse gene isolated from a cosmid library (cosmid clone cFM6.1A2) has also revealed a repeat length of about 750 base pairs. The cosmid clone contains most of the mouse filaggrin gene, but it is missing the 5'-noncoding sequences and possibly some coding sequences as well. We report here that cosmid clone cFM6.1A2 contains 20 filaggrin repeats and 15,213 base pairs of coding sequences. Sequence analysis of this clone has revealed at least two different types of repeating element. Type B has a repeat length of 750 base pairs (250 amino acids), whereas type A is 765 base pairs (255 amino acids) long and contains an additional five amino acids inserted next to an acidic sequence that delineates the amino and carboxyl termini of the filaggrin repeats. It is supposed that these additional five amino acids may alter the proteolytic sensitivity of the acidic linker sequence, thereby affecting the processing of the precursor. The random distribution of the two types of repeats in the precursor indicates that the mouse filaggrin gene arose by a complicated series of duplications and/or rearrangements.

Amino Acid Sequence↗
PubMed1989

Dermenkephalin (Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2): a potent and fully specific agonist for the delta opioid receptor.

Dermorphin, Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2 is an extraordinarily potent and highly mu-selective opioid heptapeptide isolated from amphibian skin. It is unique among peptides synthesized by animal cells in having an amino acid residue in the D-configuration. At least two different preprodermorphin cDNAs were cloned from skin of Phylomedusa sauvagei; their predicted amino acid sequences contained four to five homologous repeats of 35 amino acids, each repeat including one copy of the dermorphin progenitor sequence. Tyr-Ala-Phe-Gly-Tyr-Pro-Ser-Gly, flanked by Lys-Arg at the amino end and by Glu-Ala-Lys-Lys at the carboxyl end [Science (Wash. D. C.) 238:200-202 (1987)]. The D-Ala in position 2 in dermorphin is encoded by a usual Ala codon in the precursor sequence. Of the two prodermorphin molecules, one has a dermorphin copy replaced with a distinct heptapeptide same processing signals. Assuming the same pathway as for the release of dermorphin, processing of this precursor may yield, beside dermorphin, a copy of a new peptide, Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2. We have synthetized this peptide together with its (L-Met2)-counterpart and evaluated their respective opioid receptor selectivity in the mouse vas deferens and guinea pig ileum assays and in rat brain membrane binding assays. Overall, the data collected demonstrate that the putative prodermorphin product Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2 named dermenkephalin, behaves as a potent delta opioid agonist exhibiting high affinity and high selectivity for the delta opioid receptor. Prodermorphin, thus, offers a surprising example of an opioid biosynthetic precursor that might simultaneously generate highly potent and fully selective agonists for the mu- (morphine) and the delta (enkephalin) opioid receptors, respectively. In addition, because dermenkephalin has no structural features in common with the sequence of all the hitherto known opioid peptides, it should be a useful tool for identifying conformational determinants for high affinity and selective binding of opioids to the delta receptor.

Amino Acid Sequence↗
PubMed1985

Monoclonal proliferation of Friend murine leukemia virus-transformed myeloblastic cells occurs early in the leukemogenic process.

Integrated Friend murine leukemia virus copies were analyzed by the Southern blotting procedure in myeloblastic cell lines obtained after in vitro infection of long-term mouse bone marrow cultures. Several steps leading to the generation of malignant myeloblastic cells after a long latency period were observed in the evolution of infected cultures. Shortly after infection, a random distribution of integrated provirus copies was observed in the DNA of normally differentiating myeloid cells. In contrast, a distinct pattern of integrated Friend murine leukemia virus copies was evident in the first non-differentiating immature myeloblastic cells appearing in cultures, suggesting a monoclonal origin of these cells. For each cell line, characteristic hybridizing fragments were conserved during the 1-year culture period necessary for the acquisition of tumorigenic properties and were also observed in tumors grafted in vivo. We can conclude that monoclonality is effective very early in the myeloid transformation process, as soon as the precursor cells are blocked in their differentiation.

Animals↗
PubMed2004

Increased production of Bacillus keratinase by chromosomal integration of multiple copies of the kerA gene.

To increase the production of keratinase, stable strains of Bacillus licheniformis carrying multiple keratinase gene copies in the chromosome were developed. Integrative vectors carrying kerA with or without P43-promoter were constructed and subcloned into B. licheniformis T399D and Bacillus subtilis DB104. In T399D, multiple copies of kerA integration into the chromosome were identified and determined by Southern blot. The optimal integration of kerA was found in the range of 3-5 copies. Higher integration of gene copies (>5) caused reduced processing and secretion of the extracellular keratinase. In DB104, kerA was cloned in the plasmid, not integrated into the chromosome. The strong constitutive promoter P43 not only increased the keratinase production in plasmid-based expression in DB104 but also improved the enzyme yield of the integrants of T399D. New strains were able to enhance cell growth and enzyme yield at higher concentrations of medium substrate. When they were grown in either soy or feather medium, the keratinase activity was stable and improved by about 4-6 times.

Bacillus↗
PubMed1992

Stochastic developmental variation in the ratio of allelic rDNAs among newly differentiated, heterozygous macronuclei of Tetrahymena thermophila.

Ciliates possess nuclear dimorphism, i.e., they carry two structurally and functionally differentiated types of nuclei. The micronucleus and macronucleus serve as the germline and somatic nuclei, respectively, of the cell. The macronucleus differentiates from a mitotic sister of the micronucleus once per life cycle. Macronuclear differentiation is accompanied by a developmentally programmed set of DNA rearrangements, including chromosome fragmentation, telomere addition, and amplification. Given the diploidy of the MAC anlage, are both homologous copies of a chromosome processed and amplified equally and simultaneously in an individual differentiating MAC? We have approached this question for the case of the rDNA, exploiting previously identified DNA polymorphisms and the sensitivity of PCR. We determined allelic ratios in individual caryonide cells, i.e., the cells carrying the primary products of MAC differentiation, prior to the first division of the newly differentiated MAC. We observed stochastic variability in allelic ratios among caryonides that start with genetically identical heterozygous MACs. Either rDNA type can be in the majority. Appropriate controls make it unlikely that the ratios observed were significantly affected by variation in the assay itself. The variability may well result from the statistical variation associated with the relative timing of individual biochemical events initiating the processing and/or amplification of a few rDNA precursor molecules, presumably 4-8 at the most, in a MAC anlage. In addition to this stochastic variability, we observed a small but distinct bias in favor of the C3 rDNA. Thus the replication advantage of C3 relative to B rDNA in heterozygous MACs, previously detected during vegetative multiplication, may begin to be expressed during developmental amplification.(ABSTRACT TRUNCATED AT 250 WORDS)

Alleles↗
PubMed1991

DNA template effect on RNA splicing: two copies of the same gene in the same nucleus are processed differently.

Many cellular and viral genes are parts of complex transcription units containing multiple splicing choices. During the course of an adenoviral replicative cycle, different spliced versions of a single gene predominate, depending on the stage of infection. This is true for several adenoviral genes. In this paper we show for the viral E1B transcription unit that splice site usage regulates this process. The change in alternative splicing in this system does not depend on the sequence of the transcribed genes. Non-adenoviral genes, such as the SV40 early region and the polyoma early region, which normally show little or no regulation of spliced RNA product formation, become regulated for mRNA production after insertion into the adenoviral genome. Additional studies show that E1B splicing regulation in adenovirus is a cis effect. Staggered infections using two discernable viral genomes resulted in a situation where both early and late genomes exist in the same nucleus. Neither genome was able to impose its regulated splicing pattern on the other, indicating that the cue for the switch in viral gene splicing is not directly dependent on global changes in trans-acting splicing factors. This suggests a model where the signal for changes in RNA processing for the E1B gene is linked to the state of the DNA template or its localization within nuclear subcompartments.

Adenoviridae↗
PubMed1995

Autocatalytic gene expression occurs via transertion and membrane domain formation and underlies differentiation in bacteria: a model.

When bacteria contain two chromosomes, two or more copies of the same gene are present in the same cytoplasm and, if these copies are subject to negative regulation in trans and positive (autocatalytic) regulation in cis, one copy will be expressed at the expense of the other copy(ies). This autocatalytic process depends on the coupled transcription, or translation and insertion of nascent proteins into the membrane, or transertion. Transertion is responsible for looping genes out of the nucleoid and increasing their accessibility to transcription factors. Transertion of proteins with lipid preferences creates proteolipid domains in the membrane. These domains fuse to give two types of large domains, each associated with the expression of a particular set of genes. These large domains organize kinases, proteases and transcription factors and result in the expression of one set of genes encoding proteins with common lipid preferences from one chromosome and expression of a different set from the other. These intracellular differences underlie the production of different progeny by cell division that follows, for example, reception of extracellular signals, and that constitutes differentiation in bacteria.

Bacterial Proteins↗
PubMed2000

Modeling DNA mutation and recombination for directed evolution experiments.

Directed evolution experiments rely on the cyclical application of mutagenesis, screening and amplification in a test tube. They have led to the creation of novel proteins for a wide range of applications. However, directed evolution currently requires an uncertain, typically large, number of labor intensive and expensive experimental cycles before proteins with improved function are identified. This paper introduces predictive models for quantifying the outcome of the experiments aiding in the setup of directed evolution for maximizing the chances of obtaining DNA sequences encoding enzymes with improved activities. Two methods of DNA manipulation are analysed: error-prone PCR and DNA recombination. Error-prone PCR is a DNA replication process that intentionally introduces copying errors by imposing mutagenic reaction conditions. The proposed model calculates the probability of producing a specific nucleotide sequence after a number of PCR cycles. DNA recombination methods rely on the mixing and concatenation of genetic material from a number of parent sequences. This paper focuses on modeling a specific DNA recombination protocol, DNA shuffling. Three aspects of the DNA shuffling procedure are modeled: the fragment size distribution after random fragmentation by DNase I, the assembly of DNA fragments, and the probability of assembling specific sequences or combinations of mutations. Results obtained with the proposed models compare favorably with experimental data.

Animals↗
PubMed1998

Biochemical and kinetic analyses of NS5B RNA-dependent RNA polymerase of the hepatitis C virus.

The biochemical properties of the RNA-dependent RNA polymerase (RdRp) of the hepatitis C virus were analyzed. A hexahistidine affinity-tagged NS5B fusion protein was expressed with recombinant baculoviruses in insect cells and purified to near homogeneity. Enzymatic activity of the purified protein was inhibited by KCl or high concentrations of NaCl and was absolutely dependent on Mg2+, which could be replaced by Mn2+. NS5B was found to be processive and able to copy long heteropolymeric templates with an elongation rate of 150-200 nucleotides/min at 22 degreesC. Kinetic constants were determined for all four nucleoside triphosphates and different templates. In case of a heteropolymeric RNA template corresponding to the last 319 nucleotides of the hepatitis C virus genome, Km values for UTP, GTP, ATP, and CTP were approximately 1.0, approximately 0.5, approximately 10, and approximately 0.3 microM, respectively. The profile of several inhibitors of RdRp activity and substrate analogs indicated that the enzyme has a strong preference for ribonucleoside 5'-triphosphates and that it closely resembles 3Dpol of picornaviruses.

Hepacivirus↗
PubMed1987

Analog and digital computer theory.

Analog signals abound in the natural world. With appropriate transducers these signals can be converted to continuous voltages and can be displayed, transmitted, stored, or copied. They can be processed by analog computers, the simplest of which is an audio amplifier. With analog signals, however, there can be errors because of signals loss, interference, and noise. Binary digital signals permit only two values, either 0 ('off' or 'low') or 1 ('on' or 'high'). These signals are much less susceptible to transmission problems. Binary signals are commonly organized into 8-bit groups which can represent 256 different numbers or meanings. These data can be transmitted in either serial or parallel fashion at high rates of speed. Analog-to-digital converters permit analog signals to be transformed to digital signals. A computer consists of the memory, the processor, and the input/output devices. Memory includes the fastest registers, the very fast core memory, the peripheral storage devices such as diskettes and disks, and the very slow peripheral devices such as magnetic tape. The processor can only load and store numbers in memory, add two numbers, test a number, and provide input and output. The program counter indicates the next computer instruction to be performed. Input/output devices allow communication with the outside world and may assume many forms. A computer by itself can do nothing. A program or series of instructions is required. The most simplistic program language is assembler or machine language. Most programming is done in more sophisticated languages, however.(ABSTRACT TRUNCATED AT 250 WORDS)

Analog-Digital Conversion↗