Biomedical subjects

R Ivarie

Publications and source records attributed to R Ivarie.

At least 19 recordsLinked to original sources

PubMed2004

cDNA array analysis of Japanese quail lines divergently selected for four-week body weight.

Decades of divergent selection for 4-wk BW has produced 3 lines of growth-selected Japanese quail. P line quail have been selected for >110 generations for 4-wk posthatch BW and are nearly 3-fold larger than the randomly bred control C line. The H line has been selected for high 4-wk BW for 52 generations and the L line has been selected for low 4-wk BW for 54 generations. To identify differentially expressed genes that may play a role in defining the differences in these lines, a DNA array containing 4,704 random anonymous cDNA clones from 8-d C line embryos was screened using isotopically-labeled cDNA from the different quail lines. Array analysis yielded 3 differentially expressed cDNA clones that were confirmed by Northern blot analysis. The 35-kD quail EB1 protein, previously unidentified, was shown to have elevated transcripts in the L line and decreased transcripts in the H and P lines, compared with the C line. It was also widely expressed in embryonic and adult tissues by BLASTN analysis of chicken expressed sequence tag (EST) libraries. Two other cDNA clones are novel sequences expressed at higher levels in the L line and at lower levels in the H and P lines, one of which was more selectively expressed in embryos and adult tissues by BLASTN analysis. These limited findings suggest that anonymous cDNA array analysis is a productive means to identify differentially expressed genes in growth-selected poultry.

Animals↗

PubMed2003

Validating the hen as a bioreactor for the production of exogenous proteins in egg white.

Increased demand for the production of human biopharmaceuticals in transgenic organisms has led to an intensive effort to develop the hen as a bioreactor producing exogenous proteins in egg white via transgenesis. To date, however, robust methods for transgenic modification of the avian genome have been lacking. We have used a replication-defective retroviral vector derived from avian leukosis virus (ALV) to generate transgenic chickens expressing bacterial beta-lactamase secreted into serum and egg whites through several generations. Expression was driven by the ubiquitous cytomegalovirus (CMV) promoter. Here we describe results from a transgenic lineage (Harvey et al., 2002a,b) in which (1) the transgene was stably transmitted from a G1 founder male (5657) through several generations without silencing, (2) the protein was biologically active, and (3) the level of expression in egg whites was doubled in a G3 homozygote.

Animals↗

PubMed2002

Consistent production of transgenic chickens using replication-deficient retroviral vectors and high-throughput screening procedures.

We have developed a novel method of DNA extraction combined with a high-throughput method of gene detection allowing thousands of potentially transgenic chicks to be screened quickly and reliably. By using this method and a replication-deficient retroviral vector based on avian leukosis virus (ALV), we have demonstrated germline transmission of three different transgenes. Several generations of chickens carrying intact transgenes were produced, validating the use of the ALV retroviral vectors for large-scale production of transgenic flocks. Fourth-generation chicks that were nontransgenic, hemizygous, or homozygous for the transgene were identified with the combined genetic screening methods.

Alleles↗

PubMed2002

Expression of myostatin is not altered in lines of poultry exhibiting myofiber hyper- and hypoplasia.

Decades of selective breeding have yielded lines of poultry with substantial myofiber hyperplasia, vet little is known about what genes have been altered during the course of selection. Myostatin is a strong negative regulator of muscle mass in mice and cattle and could have been one of many genetic factors contributing to increased myofiber deposition in growth-selected lines of poultry. To test this hypothesis, the sequence and expression patterns of myostatin were analyzed in growth-selected lines of chickens and quail. The sequence of broiler myostatin cDNA, amplified via reverse transcription (RT)-PCR from embryonic muscle RNA, contained no missense mutations in the coding sequence when compared to that of White Leghorn layers, although two silent single nucleotide polymorphisms (SNP) were found. Northern analysis of myostatin transcripts from embryonic pectoralis and quadriceps showed no significant differences in expression levels between broiler and layer muscle RNA. However, levels of myostatin transcripts were greatly reduced in muscles of posthatch chicks compared to embryonic muscle. Myostatin protein was also present in broiler and layer embryonic muscle at similar levels. No significant polymorphisms or differences in RNA expression levels were found in embryonic muscles of divergently selected lines of Japanese quail. These results indicate that intense artificial selection in these growth-selected lines of poultry has neither silenced the expression of myostatin nor created null alleles via mutation in the lines analyzed.

Animal Husbandry↗

PubMed2000

A modified method of shell windowing for producing somatic or germline chimeras in fertilized chicken eggs.

Stage X chick blastoderms following oviposition were accessed via a small window in the egg. Windowing, however, substantially reduces the hatchability of eggs containing early embryos. For example, only 32 of 389 (8.2%) eggs hatched after standard windowing with or without irradiation or injection. Ex ovo culture systems can overcome this problem but are labor intensive. A modification of a standard windowing technique has yielded an average hatch rate of 32% for 892 windowed eggs independent of incubator type, gamma-irradiation, or injection of the embryo. This was a fourfold increase over a standard windowing method. Similar hatch rates were observed using fertile eggs from five chicken lines [Barred Plymouth Rock (BR), Athens-Canadian (AC), Line 0, SPAFAS, and commercial White Leghorns (WL)]. The modification involves covering the egg shell membrane with PBS after grinding away the shell and before piercing the membrane. The window is then sealed by overlaying with fresh shell membrane and cementing it in place once it has dried. The method has been used successfully for the production of somatic and germline chimeras because donor BR blastodermal cells injected into Stage X, gamma-irradiated recipient embryos from WL or AC yielded a hatch of 33.7%, of which 42.3% were feather chimeras. Two of 11 cockerels tested were germline mosaics bearing at least 1% BR sperm. The modified windowing technique may be broadly applicable in emerging technologies in avian transgenesis and development.

Animals↗

PubMed1998

An Oct-like binding factor regulates Myf-5 expression in primary avian cells.

Myogenic regulatory factors (MRFs) are hierarchical regulators of skeletal myogenesis. Many MRF promoters have been well characterized with respect to flanking sequences that control their expression. Yet the promoter elements that regulate Myf-5, the first MRF expressed during mammalian embryogenesis, are still largely unknown. Comparison of Myf-5 5' flanking regions from bovine, mouse, and chicken genes revealed three evolutionarily conserved elements proximal to the transcription start site: the TATA box, an octamer motif, termed OLS, and a 6-bp C-rich element. Mobility shift assays and DNase I footprinting analysis demonstrated that a nuclear factor(s) present in both bovine and avian muscle and nonmuscle tissues specifically recognized OLS. Furthermore, this binding activity reacted with a polyclonal Oct-1 antibody. In avian primary myoblast and fibroblast cultures, CAT reporter constructs under regulation of the proximal Myf-5 5' flanking sequence were expressed preferentially in myoblasts with CAT levels approximately 12-fold higher than in fibroblasts. The TATA box and octamer motif were important for expression in both myoblasts and fibroblasts: loss of the TATA box abolished activity, and disruption of the OLS resulted in 50-75% loss of promoter activity.

Animals↗

PubMed1994

Dexamethasone-mediated induction of MMTV-myf5 in DD3 myoblasts increases endogenous myogenin expression but does not transactivate myf5.

DD3 cells are a myoblast line generated by stable transfection of C3H10T1/2 fibroblasts with the bovine myf5 cDNA (bmyf) fused to the dexamethasone-inducible MMTV promoter. After treating proliferating cells with dexamethasone, bmyf transcripts were induced approximately fivefold within 1.5-2.5 h. Induction of bmyf was followed 4 h later by a similar increase in myogenin transcripts which was dependent on protein synthesis. The elevated level of myogenin transcripts in dividing cells was comparable to that observed in DD3 myoblasts after differentiation. The 4-h lag before the myogenin response suggested involvement of an intermediate factor. However, one possible factor, myocyte-specific enhancer binding factor (MEF-2) (a known enhancer of myogenin promoter activity) was neither detectable nor inducible by dexamethasone in proliferating cells. myoD transcripts were barely detectable and uninducible in proliferating cells but strongly upregulated during differentiation. There was also a transient twofold increase in mrf4 transcripts by dexamethasone treatment in dividing cells, while no changes were detected in the levels of Id, E12, or TnC messages. The mouse myf5 gene was silent and uninducible in DD3 cells under proliferating and differentiating conditions. We conclude that ectopic expression of MMTV-bmyf led to activation of three of the endogenous myogenic factor genes of which only myogenin showed a rapid and sustained response to dexamethasone-mediated induction of bmyf in dividing cells.

Animals↗

PubMed1994

Polyvinyl alcohol enhances detection of low abundance transcripts in early stage quail embryos in a nonradioactive whole mount in situ hybridization technique.

We have tested the ability of polyvinyl alcohol (PVA) to enhance the indoxyl-nitro blue tetrazolium alkaline phosphatase color reaction in whole mount in situ hybridizations of early stage quail embryos. Using detection of the myogenic factor qmyoD in hybridizations of 13-14 somite-pair quail embryos as a test system, 10% solutions of 13-23 kDa and 31-50 kDa PVA optimally enhanced the alkaline phosphatase color reaction. With the enhanced detection conditions, we have been able to measure qmyoD expression as early as the 3 somite-pair stage, whereas previously we were unable to detect qmyoD prior to the 10 somite-pair stage. PVA-enhanced detection of somitic qmyoD expression was specific as no corresponding signal was found in control hybridizations with a sense qmyoD probe.

Animals↗

PubMed1993

Isolation, sequence, and characterization of the bovine myogenic factor-encoding gene myf-5.

We report here the isolation, sequencing, and characterization of the bovine homologue of the myogenic factor-encoding gene myf-5 (termed bmyf). The transcription unit of the gene is 4.2 kb in length and contains two introns. Primer extension experiments mapped the transcription start point at 158 bp upstream from the predicted start codon and 29 bp downstream from a canonical TATA box. The 3' untranslated region contains four AATAAA sites, three of which are used to produce transcripts of 1.5, 2, and 3 kb in bovine fetal muscle RNA.

Amino Acid Sequence↗

PubMed1993

Expression of bovine myf5 induces ectopic skeletal muscle formation in transgenic mice.

myf5 is one of a family of four myogenic determination genes that control skeletal muscle differentiation. To study the role of myf5 in vivo, we generated transgenic mice harboring the bovine homolog, bmyf, under control of the murine sarcoma virus promoter. Ectopic expression of the full-length bmyf transgene was detected in brain and heart tissue samples of F1 progeny from transgenic founder mice. Ectopic bmyf expression activated endogenous skeletal myogenic determination genes in the hearts and brains of transgenic animals. Incomplete skeletal myogenesis in most hearts gave rise to cardiomegaly and focal areas of cardiomyopathy. In brains in which ectopic expression led to a more complete myogenesis, focal areas of multinucleated, striated myotubes containing actin, desmin, and myosin were observed. These unexpected results show that myf5 can initiate myogenic differentiation in vivo, supporting the hypothesis that myf5 is responsible for determination of cells to the myogenic lineage in normal embryogenesis.

Animals↗

PubMed1993

Delayed somite formation in a quail line exhibiting myofiber hyperplasia is accompanied by delayed expression of myogenic regulatory factors and myosin heavy chain.

A myofiber hyperplastic quail line P has been developed through selection for heavy body weight. Since the number of muscle fibers is determined early in development and skeletal muscle originates from somites, we compared somite formation and muscle-specific gene expression in P- and control C-line quail embryos. At 47 hours of incubation, C embryos had 18 somite pairs and P embryos had 14.3. By 72 and 120 hours, both lines appeared to be at the same stage of somite development. To determine whether the delay in the formation of the brachial somites was accompanied by alterations in muscle-specific gene expression, we conducted whole-mount in situ hybridization and immunofluorescence studies. At 47 hours of incubation, C embryos were expressing qmf1 in the first 12 somites, while in P embryos only the first 7 somites showed qmf1 activation. Delays in expression were also observed for qmf3 at 43 hours and for all three myogenic factors (qmf1, qmf2 and qmf3) at 60 hours. At 65 hours, C embryos expressed myosin heavy chain in the first 15 somite pairs and P embryos in the first 7. At 72 hours, the transient delay in somite formation had disappeared and there was no lag in myosin heavy chain expression between the lines. The phase delay in brachial somite formation, myogenic factors and myosin heavy chain expression may be associated with the observed myofiber hyperplasia in P-line quail by allowing an increase in the muscle stem cell population.

Animals↗

PubMed1992

The application of Markov chain analysis to oligonucleotide frequency prediction and physical mapping of Drosophila melanogaster.

Here we compare several methods for predicting oligonucleotide frequencies in 691 kb of Drosophila melanogaster DNA. As in previous work on Escherichia coli and Saccharomyces cerevisiae, a relatively simple equation based on tetranucleotide frequencies can be used in predicting frequencies of higher order oligonucleotides. For example, the mean of observed/expected abundances of 4,096 hexamers was 1.07 with a sample standard deviation of .55. This simple predictor arises by considering each base on the sense strand of D. melanogaster to depend only on the three bases 5' to it (a 3rd order Markov chain) and is more accurate than the random predictor. This equation is useful in predicting restriction enzyme fragment sizes, selecting restriction enzymes that cut preferentially in coding vs noncoding regions, and in selecting probes to fingerprint clones in contig mapping. Once again, this equation well predicts the occurrence of higher order oligonucleotides, supporting our hypothesis that this predictor holds in evolutionarily diverse organisms. When ranked from highest to lowest abundance, the observed frequencies of oligomers of a given length are closely tracked by the predicted abundances of a 3rd order Markov chain. Through use of the dependence of oligomer frequencies on base composition, we report a list of oligomers that will be useful for the completion of a cosmid physical map of D. melanogaster. Presently, the library is such that it will be possible to construct large contigs using only 30 oligonucleotide probes to fingerprint cosmids.

Animals↗

PubMed1992

Whole mount in situ detection of low abundance transcripts of the myogenic factor qmf1 and myosin heavy chain protein in quail embryos.

We describe a whole mount in situ hybridization procedure to detect and localize low abundance transcripts (qmf1) and myosin heavy chain proteins in quail embryos. The critical factor in transcript detection was the extent of proteinase K digestion. Optimal digestion increased probe accessibility and reduced background. The use of digoxigenin-labeled probes and immunological detection with anti-digoxigenin-alkaline phosphatase conjugated antibody allowed signal development in 6-10 h, unlike the 7 days required using 35S-labeled probes. Myosin heavy chain proteins were detected using immunofluorescence and confocal laser scanning microscopy to allow precise localization of signal within developing embryos.

Animals↗

PubMed1991

Prolactin-deficient GH3B3 cells are defective in the utilization of the endogenous prolactin promoter yet are fully competent to initiate transcription from a transfected prolactin promoter.

Transcription of the prolactin (PRL) gene has been analyzed in wild-type D6, PRL-deficient B3, and revertant r16 GH3 cells. Levels of processed nuclear transcripts from the PRL gene were substantially reduced in the deficient line compared to wild-type cells and returned to greater than wild-type levels in the revertant line. Rare PRL transcripts in the deficient line contained the same 5' end found on transcripts in wild-type and revertant cells as judged by primer extension and S1 nuclease protection assays, implying that the cells are deficient in utilization of the normal wild-type promoter. Deficient cells also contained wild-type levels of the PRL- and growth hormone-specific transcription factor pit-1/GHF-1, and no difference was found in the ability of extracts from wild-type and deficient cells to retard various restriction fragments from both the proximal and the distal PRL promoter regions. The deficient and wild-type cells were equally competent in initiating transcription from a transfected rat PRL promoter containing both the distal and proximal promoter elements. These observations imply that PRL-deficient cells are not defective in a trans-activating factor functioning on these PRL promoter fragments (trans model). Rather, inefficient use of the PRL promoter in the variant cells may reflect an increased methylation state of the PRL gene itself (cis model).

Animals↗

PubMed1990

A bovine homolog to the human myogenic determination factor myf-5: sequence conservation and 3' processing of transcripts.

A bovine cDNA library from fetal skeletal muscle myoblasts. was screened with a 274 bp probe to a conserved region of the mouse MyoD1 cDNA. One positive recombinant, designated bmyf, was found to contain a 1931 bp insert with an open reading frame encoding a predicted protein highly related to the human myogenic factor myf-5 Human and bovine factors are 96% homologous in their predicted amino acid sequences. At the nucleotide level, bmyf and myf-5 are 92% identical in the coding region and 74 and 80% homologous in their 5'- and 3'-untranslated regions, respectively. The bmyf cDNA, nevertheless, extends 475 nucleotides beyond a polyadenylation signal common to both cDNAs. Bmyf transcripts are expressed exclusively in skeletal muscle where three transcripts of 1.5, 2 and 3 kb were detected. While the 1.5 kb transcript lacks sequences 3' to the polyadenylation signal at nt 1415 in the bmyf cDNA, both the 2 and 3 kb RNAs contain these sequences suggesting that bmyf transcripts are alternatively polyadenylated. Bmyf cDNA can activate the expression of the myogenic program in C3H10T1/2 fibroblasts as assayed by stable and transient transfection experiments.

Amino Acid Sequence↗

PubMed1989

Synthesis of N7-ethyldeoxyguanosine 5'-triphosphate and placement of N7-ethylguanine in a specific site in a synthetic oligodeoxyribonucleotide.

N7-Ethyldeoxyguanosine 5'-triphosphate (N7-Etd-GTP) was synthesized by direct ethylation of dGTP with diethyl sulfate and purified by TLC on cellulose plates at approximately 5% yield. N7-EtdGTP was identified by its uv spectra at pH 1, 7.4, and 13, by its absorbance maxima and minima, and by the lability of the glycosidic bond to acid- and heat-induced cleavage. At pH 7.4, spontaneous cleavage of the glycosidic bond proceeded with a half-life of greater than 48 h. An enzymatic method for placing an N7-ethylguanine in a specific site in DNA was developed using terminal deoxynucleotidyltransferase and the 3' to 5' exonuclease and 5' to 3' polymerase of the Klenow fragment of Escherichia coli DNA polymerase I. The method should be readily adaptable to other modified bases as long as the modification does not occur at a base-pairing site (e.g., 5-methylcytosine, N6-methyladenine, and others).

Deoxyguanine Nucleotides↗

PubMed1989

Improved chemistry for oligodeoxyribonucleotide synthesis substantially improves restriction enzyme cleavage of a synthetic 35mer.

Two DNA duplexes of identical sequence and 35 nt in length were synthesized by an original and a highly improved version of phosphoramidite chemistry. By base composition analysis, DNA synthesized by improved chemistry (termed DMTS-imp) contained no detectable modified bases while DNA synthesized by the original chemistry (termed DMTS-std) had a large number of modifications. Under optimal reaction conditions, HhaI and RsaI cleaved the DMTS-std duplex to 76-77% completion and the DMTS-imp duplex to 96-99% completion. Restriction analysis and piperidine treatment yielded estimates of approximately 3.0% modified nucleotides in DMTS-std and approximately 1.0% in DMTS-imp. Overall, the improvements in chemistry increased the restriction efficiency of synthetic DNA up to 10-fold.

Adenine↗

PubMed1988

Mono- through hexanucleotide composition of the sense strand of yeast DNA: a Markov chain analysis.

Here we compare several methods for predicting oligonucleotide frequencies in 392 kb of yeast DNA. As in previous work on E. coli, a relatively simple equation based on tetranucleotide frequencies can be used in predicting the frequencies of longer oligonucleotides. For example, the mean of observed/expected abundances of 4,096 hexamers was 1.00 with a sample standard deviation of .18. This simple predictor arises by considering each base on the sense strand of yeast to depend only on the three bases 5' to it (a 3rd order Markov chain) and is more accurate in estimating oligonucleotide frequencies than other statistical methods examined. This equation is useful in predicting restriction enzyme fragment sizes, selecting restriction enzymes that cut preferentially in coding vs noncoding regions, and in constructing detailed physical maps of whole genomes. When ranked highest to lowest abundance, the observed frequencies of oligomers of a given length (up to 6 bases) are closely tracked by the predicted abundances of a 3rd or 4th order Markov chain. These ordered abundance curves have a power curve shape with a broad linear range with a sharp break at the top end of the curve. There is also a strong disparity between the most and least abundant oligomer with for example a 79-fold variation between the most and least abundant hexamer. The curves reveal a strong dependence of oligomer frequencies on base composition. Unlike E. Coli, there is no sharp downturn at the low end of the curves and hence, no class of oligomers rare relative to other oligomers of the same length.

Base Composition↗