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PubMed2001

Identification and expression of cosmids with an allelic variant of class I alcohol dehydrogenase in transgenic mice.

The mouse Adh1 gene exhibits tissue-specific regulation, is developmentally regulated, and is androgen regulated in kidney and adrenal tissue. To study this complex regulation phenotype a transgenic mouse approach has been used to investigate regulatory regions of the gene necessary for proper tissue expression and hormonal control. Transgenic mice have been produced with an Adh1 minigene as a reporter behind either 2.5- or 10 kb of 5'-flanking sequence [1]. Complete androgen regulation in kidney requires a region between -2.5 and -10 kb. A sequence extending to -10 kb does not confer liver expression in this minigene construct. B6.S mice express an electrophoretically variant protein resulting from a known nucleotide substitution resulting in a restriction endonuclease length polymorphism. Transgenic mice harboring B6.S cosmids can be studied for expression analysis at both protein and mRNA levels, identification of transgenic founders and inheritance studies are greatly facilitated by a PCR-restriction endonuclease cleavage approach, the entire mouse gene is used as a reporter, and the formation of heterodimeric enzyme molecules can be used to infer expression of the transgene in the proper cell types within a given tissue. Expression of a B6.S cosmid containing the entire Adh1 gene and 6 kb of 5'- and 21 kb of 3'-flanking region occurs in transgenic mice in a copy number dependent manner in a number of tissues, but expression in liver does not occur. The ability to analyze expression at the protein and mRNA levels has been confirmed using this system. Future directions will involve the use of large BAC clones modified by RARE cleavage to identify the liver specific elements necessary for expression.

Alcohol Dehydrogenase↗

PubMed2006

From famine to feast? Selecting nuclear DNA sequence loci for plant species-level phylogeny reconstruction.

Phylogenetic analyses of DNA sequences have prompted spectacular progress in assembling the Tree of Life. However, progress in constructing phylogenies among closely related species, at least for plants, has been less encouraging. We show that for plants, the rapid accumulation of DNA characters at higher taxonomic levels has not been matched by conventional sequence loci at the species level, leaving a lack of well-resolved gene trees that is hindering investigations of many fundamental questions in plant evolutionary biology. The most popular approach to address this problem has been to use low-copy nuclear genes as a source of DNA sequence data. However, this has had limited success because levels of variation among nuclear intron sequences across groups of closely related species are extremely variable and generally lower than conventionally used loci, and because no universally useful low-copy nuclear DNA sequence loci have been developed. This suggests that solutions will, for the most part, be lineage-specific, prompting a move away from 'universal' gene thinking for species-level phylogenetics. The benefits and limitations of alternative approaches to locate more variable nuclear loci are discussed and the potential of anonymous nongenic nuclear loci is highlighted. Given the virtually unlimited number of loci that can be generated using these new approaches, it is clear that effective screening will be critical for efficient selection of the most informative loci. Strategies for screening are outlined.

Cell Nucleus↗

PubMed2000

Enzyme variation and inheritance in Glechoma hederacea (Lamiaceae), a diploidized tetraploid.

The chromosome number of the polyploid species Glechoma hederacea was found to be 2n = 36 in a sample of 93 ramets derived from 27 sites in N and C Europe. Variation in 10 enzymes was surveyed in material from S Sweden and S Czech Republic. The genetic control of variation was investigated using segregating progeny from crosses and self-fertilized heterozygous plants. The genetic analysis comprised 30 of 32 putative alleles detected in the geographical survey. Five loci (Aat-2, Tpi-1, Tpi-2, Pgd-2 and Mnr) behaved as isoloci with one copy of a locus being monomorphic for a common allele, the other di-allelic for a common allele and a variant allele. In four isoloci (Pgd-1, Pgi-2, Mdh-2 and Adh), both copies of the duplicated locus were polymorphic, with one allele common to both copies and with another allele unique for each copy except for Pgd-1 where both copies were tri-allelic. Three loci, Pgm-3, Skd-1 and Skd-2 were regarded as being non-duplicated. Segregation ratios for all enzyme loci were in close agreement with expectations based on disomic inheritance. Our data suggest that the tetraploid G. hederaca is a diploidized autotetraploid.

Alleles↗

PubMed2002

Panzee, a copia-like retrotransposon from the grain legume, pigeonpea ( Cajanus cajan L.).

We report the isolation and characterization of a copia-like retrotransposon, Panzee, from pigeonpea ( Cajanus cajan). The 4947-bp Panzee element is AT rich (60%) and the integrated element is flanked by a target-site duplication of 5 bp. The structure of Panzee is that of a typical LTR-retrotransposon containing long terminal repeats (LTRs) which flank its internal region. The 5' LTR is 372 bp in length and the 3' LTR is 383 bp long. Both LTRs start with 5'-TG and end with CA-3' and have 4-bp terminal inverted repeats. The internal region between the LTRs contains two priming sites for DNA synthesis: the first, a 12-bp primer binding site complementary to initiator methionyl tRNA, is located adjacent to the 3' end of the 5' LTR and the other, a 12-bp polypurine tract lies just upstream to the 5' end of the 3' LTR. The putative polyprotein shows homology to all the proteins encoded by LTR retrotransposons, i.e. group-associated antigen ( gag), proteinase, endonuclease, reverse transcriptase (RT) and ribonuclease H (RNase H). However, the cloned copy of the element contains four frameshifts and a premature stop codon in its protein-coding domain. Genomic Southern hybridization experiments using probes derived from three different regions of the element show that Panzee or Panzee-related elements are present in high copy numbers in the pigeonpea genome. Analysis of transgenic tobacco plants containing the LTR:GUS construct shows that the 5' LTR of Panzee drives gene expression in this heterologous system in a tissue-specific manner. A phylogenetic tree constructed using reverse transcriptase sequences places Panzee in the copia group of retrotransposons.

Amino Acid Sequence↗

PubMed2004

A real-time PCR-based method to independently sample single simian immunodeficiency virus genomes from macaques with a range of viral loads.

The generation of a diverse population of viral variants is a hallmark of simian immunodeficiency virus (SIV) infection. In order to address what role this diversity plays in disease progression, accurate sampling of the viral population is necessary. However, traditional PCR-based methods often rely on amplification of multiple genomes in one reaction, leading to resampling of viral genomes and potential errors in the estimations of viral diversity, especially when sequences from only one or a small number of PCRs are examined and/or viral copy number is low. Here we describe a method to amplify one viral envelope gene per PCR, thereby avoiding resampling. For this purpose we developed a highly accurate real-time PCR method to quantify SIV copy number, then used a single SIV template in a sensitive, high-fidelity full-length envelope PCR. Using this method, we have estimated the intra-animal viral diversity for a cohort of five pig-tailed macaques (Macaca nemestrina) infected with SIVMne variants, which displayed a broad range of viral loads at setpoint.

Amino Acid Sequence↗

PubMed2001

Possible dosage effect of maternally expressed genes on visual recognition memory in Prader-Willi syndrome.

Seventeen patients with Prader-Willi syndrome (7 with paternal deletion of chromosome 15q11-q13 and 10 with maternal uniparental disomy [UPD]), and 9 controls performed a computerized visual recognition task. A series of color digital photographs were presented; most were presented twice, but the remainder appeared only once. Photographs presented twice were separated in their presentation by either 0, 10, 30, 50 or 100 intervening photographs. Subjects indicated whether each photograph had been presented previously. This procedure was implemented twice, once using photographs of foods, and once using photographs of nonfood objects. As the number of intervening photographs between the first and second presentation increased, subjects were less likely to remember having seen the photograph before. Performance by UPD subjects was less affected by increasing the number of intervening photographs relative to the other two groups, suggesting they had superior visual recognition memory. This raises the possibility of a beneficial effect of having two copies maternally expressed genes on chromosome 15. UBE3A is suggested as a possible candidate for this effect.

Adolescent↗

PubMed1996

Conservation of low-copy gene loci in Old World leishmanias identifies mechanisms of parasite evolution and diagnostic markers.

Genome plasticity has been hypothesized to be a driving force behind parasite speciation. We have evaluated divergence in single and low-copy genes in terms of locus organization, chromosomal localization and gene expression in Leishmania infantum, L. major, L. tropica and three widely divergent geographic isolates of L. donovani. Seventeen genes of low to moderate copy number (1-4 copies/haploid genome) were analyzed to identify restriction fragment length polymorphisms (RFLPs) providing heritable markers distinguishing Old World (OW) leishmanias. These RFLP markers were conserved in parasite isolates from primary infections demonstrating their utility as diagnostic tools. The species designations established by RFLP analysis of field isolates was confirmed by use of monoclonal antibodies. All 17 genes were present in each OW leishmania analyzed except LSIP (A45), which was absent from L. infantum. The 17 genes were found to be distributed among 9 distinct chromosomes. However, in spite of variations in chromosome karyotypes among the various OW leishmanias, individual gene probes localized to a similar sized chromosome from each isolate. These observations coupled with a molecular tree derived from RFLP data suggest that the OW leishmanias comprise a monophyletic lineage, with species associated with cutaneous disease exhibiting the greatest level of divergence. Data from this study supports previous observations that species causing cutaneous and visceral disease have diverged primarily by nucleotide substitutions. Such nucleotide divergence may not only lead to changes in protein function and antigenicity, but may also alter gene regulation programs as exemplified by the finding that the LdI-9-5 and LdE-6-1 genes were expressed only in visceralizing leishmanias.

Animals↗

PubMed1999

A high incidence of prophage carriage among natural isolates of Streptococcus pneumoniae.

The majority (591 of 791, or 76%) of Streptococcus pneumoniae clinical isolates examined showed the presence of two or more chromosomal SmaI fragments that hybridized with the lytA-specific DNA probe. Only one of these fragments, frequently having an approximate molecular size of 90 kb, was shown to carry the genetic determinant of the pneumococcal autolysin (N-acetylmuramic acid-L-alanine amidase). Strains carrying multiple copies of lytA homologues included both antibiotic-susceptible and -resistant isolates as well as a number of different serotypes and strains recovered from geographic sites on three continents. Mitomycin C treatment of strains carrying several lytA-hybridizing fragments caused the appearance of extrachromosomal DNA hybridizing to the lytA gene, followed by lysis of the bacteria. Such lysates contained phage particles detectable by electron microscopy. The findings suggest that the lytA-hybridizing fragments in excess of the host lytA represent components of pneumococcal bacteriophages. The high proportion of clinical isolates carrying multiple copies of lytA indicates the widespread occurrence of lysogeny, which may contribute to genetic variation in natural populations of pneumococci.

Bacteriolysis↗

PubMed1987

Insertions of up to 17 amino acids into a region of alpha-tubulin do not disrupt function in vivo.

Microtubules in yeasts are essential components of the mitotic and meiotic spindle and are necessary for nuclear movement during cell division and mating. The yeast Saccharomyces cerevisiae has two alpha-tubulin genes, TUB1 and TUB3, either of which alone is sufficient for these processes when present in a high enough copy number. Comparisons of sequences from several species reveals the presence of a variable region near the amino terminus of alpha-tubulin proteins. We perturbed the structure of this region in TUB3 by inserting into it 3, 9, or 17 amino acids and tested the ability of these altered proteins to function as the only alpha-tubulin protein in yeast cells. We found that each of these altered proteins was sufficient on its own for mitotic growth, mating, and methods of yeast. We conclude that this region can tolerate considerable variation without losing any of the highly conserved functions of alpha-tubulin. Our results suggest that variability in this region occurs because it can be tolerated, not because it specifies an important function for the protein.

Amino Acid Sequence↗

PubMed2000

Copepod development rates in relation to genome size and 18S rDNA copy number.

It is known that body sizes and temperature-independent developmental durations within two genera of calanoid Copepoda (Crustacea) are positively related to nuclear DNA contents of their somatic nuclei. Evidently because of the constraint of similar cell numbers among the species, (nucleotypic) effects of nucleus size on cell size and on cell-level processes are expressed at the whole-organism level. Here, we show that developmental durations of eight species of five genera are also negatively related to their greatly differing numbers of 18S rRNA genes per unit DNA. We propose that levels of rDNA iteration among copepods have been controlled by natural selection to regulate ribosome concentrations, therefore protein production and development rates, independently of the large variations in genome sizes, which are in turn adapted to regulating cell and therefore body sizes.

Animals↗

PubMed2004

Genetic variation in Brachyspira: chromosomal rearrangements and sequence drift distinguish B. pilosicoli from B. hyodysenteriae.

Brachyspira pilosicoli and B. hyodysenteriae are anaerobic pathogenic intestinal spirochetes differing in host range and disease manifestations. Little is known about the size, organization, or genetic content of the B. pilosicoli genome and only limited information is available regarding the genetic organization in B. hyodysenteriae. Both B. hyodysenteriae and B. pilosicoli exist as recombinant populations, and this may be due, in part, to an unusual phage-like gene transfer agent, VSH-1. To compare genetic organization in these two species, the number of mapped loci on an existing physical and genetic map of B. hyodysenteriae B78(T) was expanded, and a combined physical and genetic map of B. pilosicoli P43/6/78(T) was constructed. The B. pilosicoli genome size was about 2.5 Mb, nearly 750 kb smaller than the B. hyodysenteriae genome. Several chromosomal rearrangements have contributed to differences in the size, organization, and content of the two bacterial genomes, and such differences may influence the ability of these species to infect different hosts and cause disease. To evaluate these differences further, comparisons were focused on genes thought to contribute to host-parasite interactions. Four genetic loci (bit, fruBC, vspA, and vspH) were found in B. hyodysenteriae, but not in B. pilosicoli, while two genetic loci (clpX and mglB) were found in B. pilosicoli, but not in B. hyodysenteriae. Contrary to a previous study, an intact copy of the hlyA gene, encoding the B. hyodysenteriae beta-hemolysin gene was detected in B. pilosicoli. Although the hlyA genes of these two species were nearly identical, sequence variation was detected in the intergenic region upstream of hlyA that may alter transcription and translation efficiency of this gene in B. pilosicoli. In addition, divergence in genes flanking hlyA may affect the chemical composition of lipid attached to the mature B. pilosicoli HlyA protein resulting in reduced hemolytic activity.

Journal Article↗

PubMed2001

Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.

The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data.

Algorithms↗

PubMed1991

Molecular genetics of human cancer predisposition and progression.

The development of human cancer is generally thought to entail a series of events that cause a progressively more malignant phenotype. Such a hypothesis predicts that tumor cells of the ultimate stage will carry each of the events, cells of the penultimate stage will carry each of the events less the last one and so on. A dissection of the pathway from a normal cell to a fully malignant tumor may thus be viewed as the unraveling of a nested set of aberrations. In experiments designed to elucidate these events we have compared genotypic combinations at genomic loci defined by restriction endonuclease recognition site variation in normal and tumor tissues from patients with various forms and stages of cancer. The first step, inherited predisposition, is best described for retinoblastoma in which a recessive mutation of a locus residing in the 13q14 region of the genome is unmasked by aberrant, but specific, mitotic chromosomal segregation. Similar mechanisms involving the distal short arm of chromosome 17 are apparent in astrocytic tumors and the events are shared by cells in each malignancy state. DNA sequencing indicates that these events accomplish the homozygosis of mutant alleles of the p53 gene. Copy number amplification of the epidermal growth factor receptor gene occurs in intermediate and late-stage tumors whereas loss of heterozygosity for loci on chromosome 10 is restricted to the ultimate stage, glioblastoma multiforme. These results suggest a genetic approach to defining degrees of tumor progression and the locations of genes involved in the pathway as a prelude to their molecular isolation and characterization.

Astrocytoma↗

PubMed2000

Restricted 12p amplification and RAS mutation in human germ cell tumors of the adult testis.

Human testicular germ-cell tumors of young adults (TGCTs), both seminomas and nonseminomas, are characterized by 12p overrepresentation, mostly as isochromosomes, of which the biological and clinical significance is still unclear. A limited number of TGCTs has been identified with an additional high-level amplification of a restricted region of 12p including the K-RAS proto-oncogene. Here we show that the incidence of these restricted 12p amplifications is approximately 8% in primary TGCTs. Within a single cell formation of i(12p) and restricted 12p amplification is mutually exclusive. The borders of the amplicons cluster in short regions, and the amplicon was never found in the adjacent carcinoma in situ cells. Seminomas with the restricted 12p amplification virtually lacked apoptosis and the tumor cells showed prolonged in vitro survival like seminoma cells with a mutated RAS gene. However, no differences in proliferation index between these different groups of seminomas were found. Although patients with a seminoma containing a homogeneous restricted 12p amplification presented at a significantly younger age than those lacking it, the presence of a restricted 12p amplification/RAS mutation did not predict the stage of the disease at clinical presentation and the treatment response of primary seminomas. In 55 primary and metastatic tumors from 44 different patients who failed cisplatinum-based chemotherapy, the restricted 12p amplification and RAS mutations had the same incidence as in the consecutive series of responding patients. These data support the model that gain of 12p in TGCTs is related to invasive growth. It allows tumor cells, in particular those showing characteristics of early germ cells (ie, the seminoma cells), to survive outside their specific microenvironment. Overexpression of certain genes on 12p probably inhibits apoptosis in these tumor cells. However, the copy numbers of the restricted amplification of 12p and K-RAS mutations do not predict response to therapy and survival of the patients.

Adult↗

PubMed1994

Molecular genetic analyses of the Tyrolean Ice Man.

An approximately 5000-year-old mummified human body was recently found in the Tyrolean Alps. The DNA from tissue samples of this Late Neolithic individual, the so-called "Ice Man," has been extracted and analyzed. The number of DNA molecules surviving in the tissue was on the order of 10 genome equivalents per gram of tissue, which meant the only multi-copy sequences could be analyzed. The degradation of the DNA made the enzymatic amplification of mitochondrial DNA fragments of more than 100 to 200 base pairs difficult. One DNA sequence of a hypervariable segment of the mitochondrial control region was determined independently in two different laboratories from internal samples of the body. This sequence showed that the mitochondrial type of the Ice Man fits into the genetic variation of contemporary Europeans and that it was most closely related to mitochondrial types determined from central and northern European populations.

Animals↗

PubMed1990

The number of alleles in multigene families.

The probability distribution and moments of the number of alleles present in a sample of homologous chromosomes are studied. It is assumed that there are multiple copies of the gene on each chromosome. When there are only two copies per chromosome or when there are only two or three chromosomes, it is possible to use analytic methods to tackle the problem. Otherwise, a simulation method is suggested.

Alleles↗

PubMed1997

Roles of rDNA spacer and transcription unit-sequences in X-Y meiotic chromosome pairing in Drosophila melanogaster males.

Meiotic pairing of the X and Y chromosomes in Drosophila melanogaster males is mediated by the rDNA repeats, which are present in two tandem clusters, one in the centric X heterochromatin and the other near the base of the short arm of the Y chromosome. Deletion of the X chromosomal rDNA cluster disrupts X-Y pairing and causes high frequences of X-Y nondisjunction. Pairing can be partly restored by insertions of cloned complete rRNA genes or by rDNA fragments that include the intergenic spacer (IGS) region. A 240 bp repeated sequence in the IGS was shown to be effective in promoting pairing when present at copy numbers above five. This study further defines the rDNA sequences involved in mediating pairing. Germline insertions of a P element construct containing most of the rDNA transcription unit but no promoter or IGS region were obtained. Two single-copy insertions and four two-copy insertions proved unable to stimulate X-Y disjunction when located on an rDNA-deficient X chromosome. In addition, three insertions of a P element construct consisting of the IGS and promoter regions of the rDNA were characterized molecularly. These three insertions had previously been shown to range in pairing ability from very weak to quite strong. Molecular analysis revealed that the three insertions also vary in copy number of the 240 bp IGS repeat and that these structural differences correlate with the differences in pairing ability. These data indicate that 240 bp repeats are considerably more effective than other regions of the rDNA in stimulating chromosome pairing.

Animals↗

PubMed2002

Emergence and selection of RNA virus variants: memory and extinction.

Two features of viral quasispecies are reviewed: the presence of memory genomes as minority components of their mutant spectra, and viral extinction due to enhanced mutagenesis. Memory has been documented with several genetic markers of the important animal picornavirus foot-and-mouth disease virus (FMDV). The presence of memory genomes in viral quasispecies may accelerate their adaptive response whenever a selective constraint has already been experienced by a viral population during previous stages of its evolution. Enhanced mutagenesis has been shown to lead to losses of infectivity of a number of RNA viruses: poliovirus, vesicular stomatitis virus, human immunodeficiency virus type 1 and FMDV. These observations, based on the theoretical prediction of the existence of a copying error-threshold for maintenance of genetic information, may contribute to the development of a new antiviral strategy.

Animals↗