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A chromosome microdissection and microisolation technique in combination with filter hybridization was developed for chromosomal localization of cloned chicken genes. The DNA was obtained from microdissected chromosome regions of metaphase spreads. Dissected DNA was amplified by polymerase chain reaction (PCR). The chicken MHC gene located on the nucleolar chromosome and beta-actin gene located on chromosome 2q were chosen as tests for the procedure and then detected by dot blot analysis using amplified chromosomal DNA probed with biotinylated DNA. The study establishes the technique of using chromosome microdissection and microisolation for localization of cloned genes as a complementary or alternative approach to both in situ DNA/chromosome hybridization and fluorescent in situ hybridization.

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This study was initiated to determine whether an allelic substitution of a dominant marker gene would identify a region close to a locus affecting expression in a metric trait. The rationale for the experiment was to utilize disequilibrium between a multiple recessive randombred Rhode Island Red (RRc) stock previously selected for quantitative trait performance and an unimproved dominant marker stock (MDM). The reporter genes in the MDM were: barring (B), silver (S), creeper (Cp), rose comb (R), double uropygial gland (U), crest (Cr), dominant white (I), frizzle (F), duplex comb (D), multiple spurs (M), polydactyly (Po), blue egg (O), pea comb (P), naked neck (Na), extended black (E), white skin (W+), muffs and beard (Mb), and feathered shanks (Fsh). Appropriate reciprocal crosses of MDM with RRc and F1 to RRc produced segregating full sibs that were evaluated for association with reporter traits. Carriers of Cr, I, and F were significantly later in sexual maturity than their recessive full sibs. The pleiotropic effects of F are believed to account for the later maturity. None of the marker traits had a significant association with egg numbers or egg weight. Birds expressing E were distinctly heavier at 8 and 32 wk. There is no obvious explanation for the significant larger size for the E phenotype. The CR-I-F phenotypes were significantly smaller at 32 wk than noncarrier full sibs.

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In situ hybridization of a tritium-labeled chicken growth hormone cDNA to 62 chicken metaphase chromosome spreads was examined by analysis of silver grain distribution. A total of 554 chromosomally located grains were recorded. There was a highly significant P value for the association of silver grains to the long arm of chromosome 1. The chicken growth hormone gene can be assigned to chromosomal G-band region 1q4 because 41.3% of silver grains on chromosome 1q were located in this region.

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It has been proven that multiple cycles of metastasis can improve the metastatic potential and homing specificity of a tumor cell population. In the present study, verification of genetic alterations during changes in metastatic behavior was done by analyzing the chromosome composition of a methylcholanthrene induced murine fibrosarcoma, 3AM during multiple cycles of subcutaneous (SC) and intravenous (IV) metastasis. After 10 cycles of SC metastasis, a cell type, 7B, with a small t(19;19)(A;A) metacentric marker chromosome was enriched from 4% in the original population to 90% in FIOR. However, when the tumor cells were injected IV rather than SC, no enrichment of the 7B cell type was observed. Instead, a cell type AX with a large t(14;19)(E5;A) acrocentric marker chromosome was enriched from 1% in the parental population to 76% in F1OIV after 10 cycles of IV metastasis. The polyploid dominant FIOIV was found to be extremely high in IV metastasis (411 foci/lung) but low in SC metastasis (48 foci/lung). The diploid dominant FIOR appears to be high in both SC (163 foci/lung) and IV (301 foci/lung) metastasis. The data obtained suggest that metastasis will lead to the selection of specific preexisting cell types, and the type of cell selected will depend on the route of metastasis. Furthermore, during metastasis, new cell types may also be produced de novo through chromosomal structural and numerical aberrations.

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Chickens heterozygous for a chromosomal translocation [MN t(1;4)] were intercrossed and the progeny were analyzed for their chromosome complement. A ratio of 1 homozygous translocation carrier to 4 heterozygous translocation carriers to 1 homozygous standard chromosome carrier was noted (n = 520), rather than the 1:2:1 ratio expected from Mendelian segregation. The excess of heterozygous carriers was apparently caused by union of complementary duplication/deficient gametes. Embryonic death occurred in 68% of fertile zygotes. This finding fits very closely to expectations if alternate and adjacent meiotic disjunctions occurred at equal frequencies. As alternate disjunction frequencies increase from 0.5 to 1.0 in inter se matings, the proportion of inviable zygotes among fertilized ova will decrease from 0.625 to 0 and the proportion of translocation heterozygotes among viable progeny will decrease from 0.667 to 0.5. In instances where alternate and adjacent disjunction occur at equal frequency, preferential recovery of translocation carriers will occur. This may contribute to chromosomal diversity within a species, and possibly lead to speciation.

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The borate G-banding technique produced excellent longitudinal bands similar to trypsin G-bands on chicken chromosomes prepared from embryo material. Aged chromosome preparations on slides were pretreated in borate buffer (pH 9.2) for 10 to 30 s at 37 C, then stained in Giemsa solution. This simple G-banding procedure gave high-resolution chromosome banding and permitted the visualization of silver grains that localized cloned gene sequences or cloned DNA fragments to specific regions by in situ hybridization.

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Frozen semen is a practical means of preserving valuable germ plasm. Monitored samples of semen cryopreserved with glycerol for heterozygous, dominant marker stocks and for nine chromosomal rearrangement lines had sufficiently high fertility for germ-line retrieval. The results also indicated a potential for the genetic selection of certain lines for the freezability of spermatozoa, since stock and line differences in fertility occurred when previously frozen semen was used for insemination. Freezing the semen of stocks routinely reproduced provides insurance against possible disasters.

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It is concluded that chromatin fragments derived from irradiated chicken spermatozoa are not viable vectors for gene transfer. In three experiments conducted at sequential intervals over a period of 1 1/2 years, no marker traits were found in 1,065 G0 progeny from irradiated spermatozoa of Minnesota Dominant Marker males inseminated into recessive Rhode Island Red and White Leghorn females. The inability to secure transformants is ascribed to the following factors: a maximum of five and probably fewer potential vector fragments for each G0 progeny because of irradiation effect on spermatozoan ability to enter the germinal disc; uncertainty of DNA integrity from highly irradiated chromatin; no known mechanism for release of chromatin fragments from irradiated spermatozoa supernumerary pronuclei; and the uncertainty of selective integration into the zygotic nucleus.

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In situ DNA/chromosome hybridization techniques were used to localize the cytoplasmic beta-actin gene in the chicken. Hybridization of a beta-actin cDNA probe to metaphase chromosome spreads indicated that sequences complementary to this probe are located on the long arm of chromosome 2 (2q) and one of chromosomes 9 through 12.

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The restriction enzymes MspI, HpaII, HaeIII, and HinfI were applied in situ to chicken metaphase chromosomes to determine if they denatured specific regions to produce banding patterns. Human metaphase chromosomes were treated simultaneously to serve as controls. The MspI, HpaII, and HaeIII enzymes produced no visible banding patterns in chicken chromosomes. There was slight banding on a few chromosomes in HinfI preparations, which was similar to the occasional spontaneous banding observed in chicken chromosome preparations. The results indicate that these four enzymes do not denature C-band regions in sufficient magnitude to be detected in chicken metaphase chromosomes.

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An inherited genetic disorder causes XY embryos of the horse to develop as mares. On the basis of our study of 38 such mares, we have identified four grades or classes of XY sex reversal according to this scheme: class I, nearly normal female, of which some are fertile; class II, female with gonadal dysgenesis, normal mullerian development; class III, intersex mare with gonadal dysgenesis, abnormal mullerian development, enlarged clitoris; class IV, virilized intersex characterized by high levels of testosterone. In general, class I and class II mares were typed H-Y antigen-negative whereas class III and class IV mares were typed H-Y antigen-positive.

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The XY sex-reversal syndrome occurs when a phenotypic mare is born that has the karyotype of a stallion. The syndrome is manifested by both genotypic and phenotypic heterogeneity. The sex-reversed genetic condition occurs frequently within certain pedigrees where XY females have been found and can be readily detected by chromosome karyotyping. The phenotypic spectrum ranges from the feminine mare with a reproductive tract that is within normal limits to the greatly masculinized mare. Pedigree analysis suggests that there are two modes of inheritance: (1) an X-linked recessive or autosomal sex-limited dominant transmitted through the female and (2) an autosomal sex-limited dominant or a Y chromosomal mutation with variable expression transmitted through the male. The proportion of female to male progeny of the 69 top-producing Arabian stallions in the world is 53 to 47, indicating that the occurrence of genetic defects affecting the sex ratio such as the XY sex-reversal syndrome is relatively infrequent. In nine cases where the sex ratios of stallions deviated significantly from the expected sex ratio, the incidence of female infertility also increased. Cytogenetic screening of young animals would provide for early detection and avoid the expense and disappointment of continuous breeding attempts. Just as importantly, it would aid in the reduction and the possible elimination of this genetic condition from breeding herds.

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Some progeny resulting from interbreeding of individuals heterozygous for a pericentric inversion of chromosome 1 in the chicken have the two complementary types of recombinant chromosomes arising from a single crossing-over within the inverted segment. These individuals are capable of reproduction. Their progeny can have one or the other of the two recombinant chromosomes or, if crossing-over occurs, either a normal or an inversion chromosome.

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Colcemid at the dose level of 0.37 mg/kg/day was injected intraperitoneally to 3 sexually active chicken males for 3 consecutive days. 10-12 days after the first colcemid injection, 14-25% of the sperm population in the semen samples from the treated males was found to be diploid in DNA content by flow microfluorometric analysis. Cytogeneic and developmental analyses on early embryos indicate that, during the process of spermatogenesis, the male germ cells are most susceptible to colcemid treatment 10-12 days prior to the maturation of the spermatozoa which is equivalent to the primary through secondary spermatocyte stages in chicken males. By the application of an extremely unequal chromosomal translocation as a cytological marker of parentage, it is confirmed that the diploid sperm induced are capable of uniting with a normal haploid or diploid egg to produce a triploid or tetraploid zygote.

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A type of blindness due to lack of rods and cones in the retina was found to be controlled by a single autosomal recessive gene, rc. The mutation was first identified in the second generation descendants of a male carrying an ethyl methanesulfonate (EMS) induced chromosome translocation involving one arm of the Z sex chromosome and the long (q) arm of chromosome 3. A linkage test between the locus causing the blindness and the translocation break-point on chromosome 3 was not significant. There is no proof that the mutation was EMS induced.

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Five chromosomal rearrangements involving chromosome 1 were utilized to test linkage relationships of the blue egg--pea comb--naked neck (O-P-Na) linkage group. An F1 stock was created by crossing rearrangement carriers with individuals carrying the traits being studied. The F1 was then backcrossed to recessive, normal chromosome tester stocks. Chromosomal and phenotypic descriptions were made for each of the segregating progeny. Significant linkage relationships were shown for blue egg and pea comb on the proximal one third of the short (p) arm of chromosome 1. No linkage was found for naked neck. Barring and silver were tested against the two arms of the Z (sex) chromosome, and the linkage values suggest that these loci are on opposite arms. Blood group loci linkages between Ea-H and W4, Ea-P and Na, and Ea-A and Ea-E were confirmed. However, no linkage was observed between these loci and the marker rearrangements on the p arm of chromosome 1.

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