Destabilizing effect of chicken selection using the functional adrenal reserves criteria]

Chicken lines produced by divergent selection for the functional adrenal reserves showed significant between-line differences in the content of corticosterone and other hormones (thyroxin, progesterone), as well as in body weight, early maturation, and egg yield. DNA fingerprinting with the pGB725 probe revealed molecular changes in genomic DNA of the chicken lines subjected to plus and minus selection. The genetic distances between the original population and the selected chicken lines, which were estimated from the molecular hybridization patterns, reflected the history of breeding. Analysis of mixed DNA from several individuals of each line revealed specific hybridization bands that could serve as DNA markers during selection for the high and low corticosterone levels in blood.     

A 41–42 bp tandemly repeated sequence isolated from nuclear envelopes of chicken erythrocytes is located predominantly on microchromosomes

To study whether specific DNA sequences are associated with nuclear membranes, residual DNA was extracted from DNase-treated nuclear envelopes prepared from erythrocytes of adult chickens (Gallus domesticus). This DNA was then blunt-end ligated into a bacterial plasmid vector. DNA blot analysis and nucleotide sequence determination revealed that approximately 30% of the cloned fragments consisted of different multiples of a 41–42 bp tandemly repeated, partially symmetrical sequence. In situ hybridization to chicken chromosomes demonstrated that the sequence was located primarily on microchromosomes, although some hybridization was also observed to macrochromosomes 7 and 8. Digestion of chicken DNA with any of a number of restriction enzymes did not completely reduce the intensity of a high molecular weight band to which the repeated sequence hybridized. These results, along with those obtained from in situ hybridization, suggested that many copies of this sequence are organized into large tandem arrays, and are not dispersed in many shorter repetitive blocks throughout the chicken genome. Although the repetitive sequence constituted approximately 10% of the chicken genome, it did not hybridize to quail or turkey DNA.     

Histological Detection of Chicken δ-Crystallin DNA Sequences Introduced into Mouse Teratocarcinoma Cell Lines

In situ hybridization techniques to detect specific DNA sequences in histological sections were developed for the purpose of analyzing experimental chimeras produced by combination of mouse teratocarcinoma (TCC) cells stably carrying chicken δ-crystallin DNA sequences and normal mouse embryos. Various hybridization conditions for detection of exogenous DNA sequences were compared in samples of solid tumors of TCC lines. Of the conditions examined, denaturation of DNA in alkali and hybridization at 68°C in 6x SSC in the presence of dextran sulphate was the best for detecting δ-crystallin DNA sequences. With ³H-labelled probe under these conditions, virtually all nuclei containing more than 100 copies of chicken δ-crystallin sequences were labelled sufficiently to be distinguishable from nuclei without chicken sequences. This technique could be applied to other experimental chimeras in which specific DNA sequences can be used as markers of certain cell lineages.     

Reliability of the RNA-DNA Filter Hybridization for the Detection of Oncornavirus-Specific DNA Sequences

Denatured DNA from leukemic myeloblasts or uninfected chicken embryos, immobilized on nitrocellulose filters, was hybridized to a vast excess of [(3)H]70S RNA from purified avian myeloblastosis virus. The viral RNA was eluted from the RNA-DNA hybrids, purified, and then rehybridized in solution to an excess of either leukemic or normal chicken embryonic DNA. This study revealed that all the slow and the fast hybridizing viral RNA sequences detectable by liquid hybridization in DNA excess had hybridized to the filter bound DNA. Both techniques also gave similar values for the number of 28S ribosomal RNA genes contained in a chicken cell genome: 210 by the liquid hybridization procedure and 218 by the filter hybridization technique. Therefore, filter hybridization can accurately detect DNA sequences present in relatively few numbers in the genome of higher organisms.     

Genetic characterization of highly inbred chicken lines by two DNA methods: DNA fingerprinting and polymerase chain reaction using arbitrary primers

Thirteen highly inbred chicken lines were analysed at the DNA level by DNA fingerprinting (DFP) and by polymerase chain reaction (PCR) using random primers. In general, the DFP patterns of individuals within a line were identical. The DFP band-sharing (BS) values among lines from different breeds (Leghorn, Fayoumi, Spanish) ranged from 0.10 to 0.20. The DFP BS values among Leghorn lines from different genetic backgrounds ranged from 0.42 to 0.79. The DFP BS values among lines selected for different major histocompatibility complex serotypes from a common genetic background ranged from 0.70 to 0.95. Some randomly amplified polymorphic DNA (RAPD) PCR products were specific to a single line, some to all lines from the same genetic base, and some to all lines from the same breed. The RAPD-PCR band-sharing values ranged from 0.66 to 0.99 for all between-line comparisons. Thus, the ability to detect biodiversity at the DNA level was greater in this study for DFP than for RAPD-PCR. The possible origin of line-specific bands, relative advantages of detecting biodiversity by using different molecular screening techniques and uses of highly inbred chicken lines in molecular analysis are discussed.     

The distribution of endogenous chicken retrovirus sequences in the DNA of galliform birds does not coincide with avian phylogenetic relationships.

The chicken is a domesticated form of Red Jungle-fowl (Gallus gallus), which belongs to the Pheasant family (Phasianidae) within the order Galliformes. Domestic chickens carry the genome of the endogenous retrovirus RAV-O as DNA sequences integrated into host chromosomes transmitted through the germ line. We have examined the presence and distribution of RAV-O-related sequences in the DNA of Red Junglefowl and other closely related species of Junglefowl, as well as more distantly related Pheasants and Quail. DNA sequences homologous to RAV-O were analyzed by molecular hybridization in liquid and after electrophoresis of restriction endonuclease fragments. The presence of RAV-O-related sequences in avian DNA does not correlate with phylogenetic relationships. Under stringent conditions of hybridization in liquid, DNA sequences homologous to RAV-O cDNA were detected at high levels (greater than 80% homology( only in the genomes of the domestic chicken and its phylogenetic ancestor, the Red Junglefowl (Gallus gallus). The DNA of two other species of Gallus (G. sonnerati, Sonnerat's Junglefowl and G. varius, Green Junglefowl), of Ring-necked Pheasant and of Japanese Quail contained sequences with less than 10% homology to RAV-O cDNA. Under conditions permitting mismatching, however, Ring-necked Pheasant DNA hybridized up to 50% of the RAV-O cDNA, and Quail DNA 24%, whereas the extent of hybridization to Sonnerat's and Green Junglefowl DNA was not markedly increased. Analysis of restriction enzyme digests revealed several distinct fragments of DNA hybridizing to chick retrovirus cDNA in both Red Junglefowl and domestic chicken, and multiple fragments in DNA from two species of Phasianus. No fragments with sequences related to chicken retroviruses were found, however, in digests of DNA prepared from Sonnerat's, Ceylonese and Green Junglefowl, from two other Pheasant genera (Chrysolophus and Lophura), or from one Quail genus (Coturnix). Thus the DNA of three Junglefowl species closely related to Gallus gallus lacked RAV-O sequences while the DNA of more distantly related Phasianus species showed significant homology. These results show that RAV-O-related sequences have not diverged together with the normal host genes during the evolution of the Phasianidae. Although RAV-O sequences are endogenous in all domestic chickens and Red Junglefowl studied thus far, it appears that the RAV-O genome has been introduced relatively recently into the germ line of Gallus gallus, following speciation but before domestication, and independently of the related sequences found in members of the genus Phasianus.     

DNA amplification in multidrug, cross-resistant Chinese hamster ovary cells: molecular characterization and cytogenetic localization of the amplified DNA

Vincristine-resistant (VCR) Chinese hamster ovary (CHO) cells have been established by stepwise selection in increasing concentrations of vincristine. These cells exhibit multidrug cross-resistance to a number of drugs that have no structural or functional similarities. Cytogenetic analyses of resistant cells revealed the presence of double minutes and expanded chromosomal segments, thus implicating gene amplification as a possible mechanism of resistance. An amplified DNA segment isolated from other multidrug cross-resistant CHO cell lines (Roninson, I. B., H. T. Abelson, D. E. Housman, N. Howell, and A. Varshavsky, 1984, Nature (Lond.), 309:626-628) is also amplified in our VCR lines. This DNA segment was used as a probe to screen a cosmid library of VCR genomic DNA, and overlapping clones were retrieved. All of these segments, totaling approximately 45 kilobases (kb), were amplified in VCR cells. Using in situ hybridization, we localized the amplification domain to the long arm of CHO chromosome 1 or Z1. Northern hybridization analysis revealed that a 4.3-kb mRNA was encoded by this amplified DNA domain and was over-produced in the VCR cells. Suggestions for the involvement of these amplified DNA segments in the acquisition of multidrug cross-resistance in animal cells are also presented.     

Babesia bovis: molecular and biological characteristics of cloned parasite lines

An in vivo limiting dilution technique was used to produce several Babesia bovis cloned lines with which to study the basis of virulence and immunogenicity in this parasite. DNA hybridization using a cloned DNA fragment from the BabR locus demonstrated that the cloned lines were a more restricted genetic population than the parent strain. Biosynthetic labeling and immunoprecipitation studies indicated that the cloned lines differed from each other and from the parentals in the expression of a small number of polypeptides and antigens. Animal trials with three of the lines demonstrated that the parental line contains both virulent and avirulent parasite populations, at least three of which are not tick transmissible, and that while the lines do provide significant protection against heterologous challenge, they may not give as effective protection as the parental line. These experiments demonstrated the existence of subpopulations with distinctive molecular and biological properties, providing evidence that the attenuation process is based on the selection of preexisting parasite subpopulations combined with the ability of these parasites to vary genetically.     

Chicken serum inhibits lectin-induced proliferation of autologous splenic mononuclear cells

Splenic mononuclear cells (SMC) proliferate well in response to phytomitogens in serum-free medium. Low concentrations of corticosterone significantly inhibit both spontaneous (1 ng/ml) and phytohemagglutinin-induced proliferation (12 ng/ml) of chicken SMC. Addition of as little as 3% normal heat-inactivated chicken serum to both autologous and heterologous suspensions of chicken SMC causes a 50% inhibition in lectin-induced proliferation. This suppression by normal chicken serum increases in a dose-dependent manner. Inhibition of DNA synthesis by chicken serum is not due to endogenous corticosterone because charcoal stripping of the serum to deplete glucocorticoids does not remove the immunosuppression. Chicken serum is not cytotoxic to lymphoid cells. These data demonstrate that low, physiologic concentrations of corticosterone inhibit proliferation of chicken SMC. Utilization of a serum-free culture system also permitted the identification of a serum factor that appears to be even more immunosuppressive than corticosterone. Since this factor inhibits the lectin-induced proliferation of both autologous and heterologous avian lymphoid cells, it may be a very important naturally occurring immunoregulatory compound.     

Hybridization of Rous Sarcoma Virus Deoxyribonucleic Acid Polymerase Product and Ribonucleic Acids from Chicken and Rat Cells Infected with Rous Sarcoma Virus

Rous sarcoma virus (RSV)-specific ribonucleic acid (RNA) in virus-producing chicken cells and non-virus-producing rat cells infected with RSV was studied by hybridization with the endogenous deoxyribonucleic acid (DNA) product of the RSV virion DNA polymerase system. By hybridizing the total DNA product with excess virion RNA, the product DNA was separated into hybridized (“minus”) and nonhybridized (“plus”) DNA. The “minus” DNA was complementary to at least 20% of the RNA from RSV which remained of high molecular weight after denaturation. A maximum of approximately 65% hybridization was observed between “minus” DNA and RSV RNA or RSV-infected chicken cell RNA. A maximum of about 60% hybridization was observed between “minus” DNA and RSV-infected rat cell RNA. RSV-infected chicken cells contained RSV-specific RNA equivalent to about 6,000 virions per cell. RSV-infected rat cells contained RSV-specific RNA equivalent to approximately 400 virions per cell. Neither cell type contained detectable RNA complementary to virion RNA. The RSV-specific RNA in RSV-infected rat cells did not appear to be qualitatively different from that in RSV-infected chicken cells.     

Analysis of DNA methylation polymorphism in a set of stable chromosome translocation lines

Interspecific hybridization is associated with the origin of novel traits and confers increased vigor compared with the parent lines, although its molecular basis is poorly understood. We report here the identification of genetic and epigenetic changes in a set of wheat–rye translocation lines (R59, R57, and R25) which exhibited novel heritable morphological characteristics compared with the parent lines (MY11 and L155). Genome in situ hybridization and amplified fragment length polymorphism analyses revealed no obvious variations in the primary structure of the genome in different translocation lines, with the exception of the same 1RS chromosome translocation. Global assessment of the extent and pattern of cytosine methylation alterations by methylation-sensitive amplified polymorphism (MSAP) analyses revealed differences in the extent of genomic DNA methylation between the rye and wheat parent lines. Fully-methylated sites were significantly increased and hemi-methylated sites were markedly decreased in the genome of translocation lines compared with the wheat parental cultivar MY11. Comparisons of different MSAP patterns revealed both monomorphic and polymorphic sites between translocation lines and wheat parents. Sequencing of 44 isolated fragments that showed methylation alterations indicated that cellular genes and especially transposable elements were targets for methylation alterations in translocation lines. The present study provides further understanding of the rules governing the distribution and existence of DNA methylation variations induced in the wheat genome during alien germplasm introduction. Furthermore, our study provides insights into the relationship between DNA methylation and hybrid vigor as well as a theoretical basis for further fundamental research and breeding application.     

Latent Marek's disease virus can be activated from its chromosomally integrated state in herpesvirus-transformed lymphoma cells.

Marek's disease virus (MDV), a lymphotropic herpesvirus, induces T-cell lymphomas in chicken, its natural host. The lymphoma cells are latently infected with MDV but the viral contribution to the transformed phenotype is not understood. To investigate the virus-cell interaction, we focused on the status of MDV in the transformed cells. By the use of highly sensitive fluorescent in situ hybridization with metaphase chromosomes, we found (i) MDV DNA to be randomly integrated at multiple sites in the chromosomes of primary lymphoma cells from chicken tissues; (ii) extrachromosomal, circular MDV genomes were absent and linear virion DNA was usually not detectable in the latently infected lymphoma cells; (iii) the pattern of integration sites revealed the clonal origin of the tumour cells; which (iv) was retained in in vitro established cell lines derived from primary lymphomas; (v) activation of the lytic phase of MDV's life cycle occurred in vitro suggesting that MDV can escape from its integrated status by an unknown mechanism.     

Molecular cloning and characterization of the chicken gene homologous to the transforming gene of rous sarcoma virus

Four molecular clones containing DNA homologous to the Rous sarcoma virus transforming gene (src) have been isolated from a random library of normal chicken DNA. The four clones are distinct overlapping isolates, which together span approximately 33 kb of cellular DNA. The cloned locus appears to represent the major region of chicken DNA homologous to src, since src-containing restriction fragments of this locus account for the fragments detected by hybridization of src-specific probe to restriction digests of total chicken DNA. Analysis of the cloned chicken src locus by restriction and heteroduplex mapping indicates that the locus contains 1.6-1.9 kb of DNA homologous to the viral src gene. The chicken DNA sequences homologous to viral src are interrupted by five or six nonhomologous regions, totaling approximately 6 kb, which presumably represent introns in the cellular src gene.     

Unique sequence, ski, in Sloan-Kettering avian retroviruses with properties of a new cell-derived oncogene.

The Sloan-Kettering viruses (SKVs) are a group of transforming retroviruses that were isolated from chicken embryo cells which had been infected with the avian leukosis virus transformation-defective Bratislava 77 (tdB77). Each of the SKV isolates was shown to contain multiple genomes of different sizes indicating the presence of several viruses in addition to tdB77. To identify and characterize the putative transforming gene(s) of the SKVs, we used hybridization selection to isolate the fraction of a representative cDNA which was SKV specific. Both solution and blot hybridization studies with viral RNAs showed that the specific probe contained a sequence, ski, that was at least partially held in common by the multiple SKV genomes. This conclusion was confirmed by the observation that a molecularly cloned ski probe also hybridized to each of the multiple SKV genomes. Southern blots of chicken DNA revealed homologs of ski (c-ski) which were not associated with endogenous viral loci. Results showing that c-ski was expressed in polyadenylated cytoplasmic RNA of uninfected chicken cells indicated that it is a functional gene. Other data showed that c-ski was conserved in avian and mammalian evolution, suggesting a functional role for the gene in species other than chickens. Using ski cDNA in solution hybridizations with viral RNAs and in Southern blot hybridization with cloned retroviral oncogenes, we did not detect any relationship between ski and any of 15 previously identified oncogenes.     

Sequence polymorphisms, allelic expression status and chromosome locations of the chicken IGF2 and MPR1 genes

By screening 26 chicken breeds and lines, DNA polymorphisms were identified in the IGF2 and MPR1 genes, of which mammalian homologues are parentally imprinted, and the GAPD gene, a housekeeping control. Using the polymorphisms as genetic markers, we found that all three genes are expressed biallelically in embryonic tissues. IGF2 and MPR1 were mapped on chicken chromosomes 5 and 3, respectively, by fluorescence in situ hybridization, demonstrating conserved linkage homology between mammals and birds.     

Contact-dependent regulation of vinculin expression in cultured fibroblasts: a study with vinculin-specific cDNA probes.

Vinculin specific cDNA clones were isolated from chicken embryo fibroblast (CEF) cDNA library in lambda gt11. The clones, ranging in size from 2.8 to 5.0 kb, were initially selected by rabbit antibodies to vinculin. Their identity was further confirmed by their specific reactivities with a battery of different vinculin-specific monoclonal antibodies. Southern blot analysis of restriction enzyme digested chicken spleen DNA suggested that all the isolated cDNA clones correspond to the same gene(s). Northern blot hybridization revealed that the vinculin-specific cDNA clones react with a single 6.5 kb mRNA in total cellular RNA preparations of CEF, whole chicken embryos and chicken gizzard smooth muscle. Moreover, fractionation of CEF poly(A)+ RNA by sucrose gradient centrifugation followed by translation in cell free system indicated that the mRNA coding for vinculin has a size of about 6.0-7.0 kb. The identity of these clones was finally confirmed by selection hybridization assay. The isolated vinculin-specific cDNA probes were subsequently used in order to study the effect of substrate adhesiveness on the expression of vinculin. We show here that cells cultured on highly adhesive substrate, such as endothelial extracellular matrix (ECM), form large vinculin-rich focal contacts, while cells grown on poorly adhesive substrate poly(2-hydroxyethyl methacrylate) [poly(HEMA)] contain only small distorted vinculin spots. These morphological differences were accompanied by over 5-fold reduction in vinculin synthesis in cells growing on poly(HEMA), compared to those cultured on the ECM and over 7.5-fold decrease in the levels of vinculin-specific mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of commercially valuable traits in hexaploid triticale lines with Aegilops introgressions as dependent on the genome composition

Introgressive hybridization is an efficient means to improve the genetic diversity of cultivated cereals, including triticale. To identify the triticale lines with Aegilops introgressions, genotyping was carried out with ten lines obtained by crossing hexaploid triticale with genome-substitution forms of the common wheat cultivar Avrora: Avrolata (AABBUU), Avrodes (AABBSS), and Avrotika (AABBTT). The genome composition of the triticale lines was studied by in situ hybridization, and recombination events involving Aegilops and/or common wheat chromosomes were assumed for nine out of the ten lines. Translocations involving rye chromosomes were not observed. Substitutions for rye chromosomes were detected in two lines resulting from crosses with Avrolata. Genomic in situ hybridization (GISH) with Ae. umbellulata DNA and molecular genetic analysis showed that chromosome 1R was substituted with Ae. umbellulata chromosome 1U in one of the lines and that 2R(2U) substitution took place in the other line. Fluorescence in situ hybridization (FISH) with the Spelt 1 and pSc119.2 probes revealed a translocation from Ae. speltoides to the long arm of chromosome 1B in one of the two lines resulting from crosses with Avrodes and a translocation in the long arm of chromosome 7B in the other line. In addition, the pSc119.2 probe revealed chromosome 1B rearrangements in four lines resulting from crosses with Avrolata and in a line resulting from crosses with Avrotika. The lines were tested for main productivity parameters. A negative effect on all productivity parameters was demonstrated for Ae. umbellulata chromosome 2U. The overwinter survival in all of the lines was similar to or even higher than in the original triticale cultivars. A substantial increase in winter resistance as compared with the parental cultivar was observed for the line carrying the T7BS-7SL translocation. The line with the 1R(1U) chromosome substitution seemed promising for the baking properties of triticale.     

Chicken microsatellite markers isolated from libraries enriched for simple tandem repeats

The total number of microsatellite loci is considered to be at least 10-fold lower in avian species than in mammalian species. Therefore, efficient large-scale cloning of chicken microsatellites, as required for the construction of a high-resolution linkage map, is facilitated by the construction of libraries using an enrichment strategy. In this study, a plasmid library enriched for tandem repeats was constructed from chicken genomic DNA by hybridization selection. Using this technique the proportion of recombinant clones that cross-hybridized to probes containing simple tandem repeats was raised to 16%, compared with < 0·1% in a non-enriched library. Primers were designed from 121 different sequences. Polymerase chain reaction (PCR) analysis of two chicken reference pedigrees enabled 72 loci to be localized within the collaborative chicken genetic map, and at least 30 of the remaining loci have been shown to be informative in these or other crosses.