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A genome-wide scan was performed in order to identify Quantitative Trait Loci (QTL) associated with growth in a population segregating high growth (hg), a partially recessive mutation that enhances growth rate and body size in the mouse. A sample of 262 hg/hg mice was selected from a C57BL/6J-hg/hg x CAST/EiJ F2 cross and typed with 79 SSLP markers distributed across the genome. Eight significant loci were identified through interval mapping. Loci on Chromosomes (Chrs) 2 and 8 affected the growth rate of F2 mice. Loci on Chr 2 and 11 affected growth rate and carcass lean mass (protein and ash). A locus on Chr 9 modified femur length and another one in Chr 17 affected both carcass lean mass and femur length, but none of these had significant effects on growth rate. Loci on Chrs 5 and 9 modified carcass fat content. Additive effects were positive for C57BL/6J alleles, except for the two loci affecting carcass fatness. Typing of selected markers in 274 +/+ F2 mice revealed significant interactions between hg and other growth QTL, which were detected as changes in gene action (additive or dominant) and in allele substitution effects. Knowledge about interactions between loci, especially when major genes are involved, will help in the identification of positional candidate genes and in the understanding of the complex genetic regulation of growth rate and body size in mammals.

This work describes the construction and characterization of a sequence characterized amplified DNA region (SCAR DNA), designated OmyP9, that was derived from a RAPD marker associated with the sex chromosomes in rainbow trout. A RsaI restriction fragment length polymorphism in OmyP9 identifies variants A, B and C. We found six OmyP9 variant phenotypes - A, B, C, AB, BC and ABC, in 186 individuals of seven different rainbow trout strains. The patterns of inheritance of OmyP9 in 139 fingerlings from 10 crosses of three strains of rainbow trout were studied. The males had a greater representation of the A variant (93.3%) suggesting an association with the Y chromosome. All male fingerlings analysed inherited the A variant from their male parents. These results support the hypothesis that OmyP9 is located on the sex chromosomes of rainbow trout, and that for the males studied the A variant is located on the Y chromosome in a region close to sex determinants and/or in a sector where the genetic recombination between X and Y is restricted. The present evidence also supports our previous hypothesis that OmyP9 is organized as a tandem repeated sequence in the sex chromosomes of rainbow trout. We feel that the OmyP9 RsaI marker can be used for sex identification in crosses where it is possible to determine the phenotype of the parents.

The attainment of a specific mature body size is one of the most fundamental differences among species of mammals. Moreover, body size seems to be the central factor underlying differences in traits such as growth rate, energy metabolism and body composition. An important proportion of this variability is of genetic origin. The goal of the genetic analysis of animal growth is to understand its "genetic architecture", that is the number and position of loci affecting the trait, the magnitude of their effects, allele frequencies and types of gene action. In this review, the different strategies developed to identify and characterize genes involved in the regulation of growth in the mouse are described, with emphasis on the methods developed to map loci contributing to the regulation of quantitative traits (QTLs).

Characterizing causal molecular defects in mouse models of overgrowth or dwarfism helps to identify the key genes and pathways that regulate the growth process. We report here the molecular basis for high growth (hg), a spontaneous mutation that causes a 30-50% increase in postnatal growth. We conclude that hg is an allele of the suppressor of cytokine signaling 2 (Socs2), a member of a family of regulators of cytokine signal transduction. We demonstrate mapping of Socs2 to the hg region, lack of Socs2 mRNA expression, a disruption of the Socs2 locus in high-growth (HG) mice, and a similarity of phenotypes of HG mice and Socs2(-/-) mice generated by gene targeting. Characteristics of the HG phenotype suggest that Socs2 deficiency affects growth prenatally and postnatally most likely through deregulating the growth hormone (GH)/insulin-like growth factor I (IGF1). These results demonstrate a critical role for Socs2 in controlling growth., (Copyright 2001 Academic Press.)

Nongenetic factors such as nutrition modulate the effects of genes responsible for overgrowth in animals. The goal of this study was to examine the importance of genotype x diet interactions on the effects of a major locus that regulates growth in the mouse. We have examined the phenotype of high growth (hg), a partially recessive autosomal locus that increases growth rate and mature body size. C57BL/6J (C57) and congenic C57BL/6J-hg/hg (HG) mice were fed three experimental diets differing in protein and energy content from 3 to 12 wk of age. HG mice grew faster and were, on average, 51% heavier than C57 at 12 wk of age. Feed intake was higher in HG mice but proportional to the increase in body weight. The magnitude of the differences in body size and composition between lines depended on the interaction between genotype and the protein/energy ratio of the diet. In C57, the diets modified the level of fatness without changing adult lean mass. However, in HG the diets differentially affected both linear growth and body composition. In general, HG had higher plasma levels of insulin-like growth factor I at 3 and 12 wk than C57. Plasma insulin did not differ between lines, but leptin was higher for C57 mice fed a high-energy diet. These results show that the effects of hg on growth are modulated by diet composition. Therefore, this mutation could be a valuable model with which to study the genetic and nutritional aspects of overgrowth disorders.

Reproductive performance was characterized in females of a line of mice homozygous for a recessive mutation with a major effect on growth, the high growth (hg) mutation. Specifically, the age at onset of puberty, length of the estrous cycle, ovulation rate, and gestation length in high growth (HG) mice were determined and compared with control mice. HG females reached puberty at an age similar to control animals, exhibited normal ovulation rates and estrous cycle length, but had a shorter length of estrus. Number of fetuses at Day 17 of gestation was greater and gestation length was longer in HG females. The HG females exhibited a variety of reproductive disorders, including increased incidence of repeated detection of mating plugs without intervening pregnancy ("replug"), increased incidence of repeated detection of mating plugs within an interval of 1-8 days without intervening pregnancy ("recycle"), increased interval between pairing with a male and detection of the first mating plug, increased interval between mating and conception, increased postnatal mortality, decreased litter size, and increased incidence of vaginal septum.

The most common kappa casein (kappa-CN) variants, kappa-CN A and kappa-CN B, are synthesised differentially in the lactating mammary gland of heterozygous animals (kappa-CN AB). In this study we evaluated several approaches for quantification of allele specific mRNA transcripts. The most consistent results were obtained using allele specific RT-PCR and capillary electrophoresis. On average, 13.4% more allele B specific than A specific transcripts were found. DNA sequencing of the proximal promoter region in several homozygous animals (kappa-CN AA, BB, EE) did not reveal any allele specific polymorphisms. Using the EMSA and DNase I footprinting we confirmed functional binding sites for three transcription factors (AP-2, NF1 and MGF) within the kappa-CN proximal promoter region. Sequence analysis of the 3'-UTR of the kappa-CN gene revealed seven allele specific sites. Two of these allelic differences were close to previously identified 3'-end regulatory sequences. In addition, allele specific differences in length between mRNAs of both variants were found. The two later findings suggest a possible post translational control determining content differences of kappa-CN in milk.

A null beta-casein allele (CSN2O) was investigated in Creole and Pyrenean goats producing milk devoid of beta-casein (CSN2). Northern blot analyses of total mammary RNA showed much lower amounts of CSN2 transcripts that were similar in size to the wild-type counterpart. The amount of CSN2O mRNA was roughly 5% of the amount of mRNA obtained at the same age and stage of lactation from CSN2A/A goats. Comparative sequence analyses of full-length CSN2O and CSN2A cDNAs showed that both alleles were of similar size, but allele CSN2O had a one-nucleotide deletion in the 5' end of exon 7, which introduces a premature stop codon. The open reading frame of allele CSN2O encodes a shortened polypeptide of 72 amino acids, compared to 223 amino acids for caprine pre beta-casein A. Comparative analyses of RT-PCR products suggested that alleles CSN2O and CSN2A might also differ in the amount and relative ratio of minor deleted CSN2 transcripts. The lower amount of CSN2O mRNA was associated with the occurrence of the premature stop codon which may mediate a rapid decay of CSN2O mRNA and promote skipping of nucleotide stretches containing premature nonsense triplets.

Quantification of beta-lactoglobulin A and B in the milk of heterozygous animals has revealed a differential content of these two variants. Nucleotide sequence analysis of the first 733 bp of the bovine beta-lactoglobulin promoter has indicated the presence of ten polymorphic sites, nine of them being allele A and B specific mutations. To study the differential expression of these alleles, constructs containing 753 bp long fragments of the bovine beta-lactoglobulin A and B associated promoters were used in transient transfection experiments in HC11 cells. A differential transcription activity directed by the A and B specific promoters was consistently observed. The relative expression levels were 57% for beta-lactoglobulin A and 43% for beta-lactoglobulin B promoters. An allele-specific mutation has been reported to have a differential binding affinity to the activator protein-2 between the beta-lactoglobulin A and B promoters. However, experiments in HC11 cells where the activator protein-2 binding sites were mutated in both beta-lactoglobulin A and B promoters showed no major differences in activity between the mutated and native promoters.

Embryonic stem (ES) cells are pluripotent cells isolated from in vitro culture of preimplantation embryos. Experiments were undertaken to identify preimplantation embryonic stages and culture conditions under which pluripotent, porcine embryo-derived cell lines could be isolated. Cell lines were established from in vitro culture of intact, porcine early hatched blastocysts and isolated inner cell masses (ICM) from intermediate and late hatched blastocysts on feeder layers prepared from permanent mouse embryonic fibroblasts (STO). The cells of these porcine embryo-derived cell lines had a morphology similar to that of murine ES cells, but colony morphology was more epithelial-like. The cell lines retained a normal diploid karyotype, consistently expressed alkaline phosphatase activity, and survived cryopreservation. When subjected to in vitro differentiation, either spontaneous or induced, the embryo-derived cell lines differentiated extensively into a wide range of cell types representing the 3 embryonic germ layers. In vivo pluripotency of the cells was demonstrated by birth of a chimeric piglet, documented by pigmentation and DNA markers, and the ability to direct the development of nuclear-transfer embryos to the blastocyst stage. Such pluripotent embryo-derived cells provide a potential route for porcine genetic manipulation.

Animals with extreme body growth frequently experience poor reproductive performance, but the cause for this association has not been clearly established. A line of mice homozygous for the high growth (hg) mutation, a mutation that has a major effect on post-weaning growth, exhibits several reproductive deficits including an abnormally high incidence of luteal failure. The objective of the present study was to investigate luteal failure in high-growth (HG) mice during pregnancy and to determine whether the cause of the apparent luteal failure resides in the ovary or the hypothalamic-pituitary unit. In HG females with a demonstrated history of infertility, progesterone injections (1 mg s.c. daily) beginning on Day 1 postcoitum (p.c.) increased the proportion of animals pregnant at Day 17 of gestation. Twice-daily injections of 100 microgram of ovine prolactin (PRL) in alkaline saline given to another group of females beginning on Day 1 p.c. increased the proportion of HG females that were pregnant on Day 6 of gestation compared with saline-injected HG females, although PRL did not increase the pregnancy rate in HG females when compared with a group of noninjected control females. When ovaries from HG females were transplanted into ovariectomized congenic C57 hosts, the C57 graft hosts displayed normal estrous cycles, and upon mating the majority of graft hosts became pregnant. In contrast, when ovaries from C57 females were transplanted into ovariectomized HG hosts, the HG graft hosts displayed normal estrous cycles, but upon mating most were unable to maintain pregnancy. These results suggest that the hypothalamic-pituitary unit of the HG female provides an inadequate signal to the ovaries to maintain pregnancy. Luteal failure in HG females may be due to insufficient PRL as required for establishment and early maintenance of the CL during pregnancy in mice.

The mouse high growth (hg) gene was identified in a selection experiment for rapid growth. It produces a 30-50% increase in weight gain of homozygous individuals without resulting in obesity. Recently, hg was mapped to a deletion around marker D10Mit69. Here we report a map of yeast artificial chromosome and bacterial artificial chromosome clones spanning the entire region deleted in high-growth mice. The size of the deletion estimated from the clone lengths is approximately 500 kb. Using exon trapping, the murine Raidd/Cradd gene was identified in the hg region, and its cDNA sequence and expression pattern were determined. The human RAIDD/CRADD was mapped using radiation hybrid mapping to human chromosome 12, 2.9 cR distal to marker AFMB311WC5. The identified Raidd/Cradd gene, which is deleted in high-growth mice, presents a potential candidate for hg., (Copyright 1998 Academic Press.)

The objectives of this study were to form appropriate composite kappa-beta-alpha(s1)-CN genotypes in order to assess which genotypes were favorably associated with first lactation milk production, fat and protein percentages, and fat and protein production for data of 916 Holstein and 116 Jersey cows. Multiple-trait animal models were used with assumed fixed effects for herd, year and season of calving, age at calving, days open, composite kappa-beta-alpha(s1)-CN genotypes, and beta-LG genotypes. The differences between the beta-LG genotypes for production traits were not statistically significant for either breed. The proportion of phenotypic variance that was due to the composite kappa-beta-alpha(s1)-Cn genotypes was 5% for milk production, 4% for protein production, and 3% for fat percentage. The kappa-beta-alpha(s1)-CN genotype ABA1A2BB was superior to the comparable AAA1A2BB and ABA1A1BB genotypes by 252 and 338 kg for first lactation milk yield, respectively, and 8.7 and 11.5 kg for protein yield, respectively. Thus, neither the beta-CN A2 allele nor the kappa-CN B allele alone had a positive effect on milk and protein production, but the joint effect was strongly positive. These results may be explained by epistatic effects between the kappa-CN and beta-CN loci and possibly by closely linked quantitative trait loci with favorable alleles on the kappa-beta-alpha(s1)-CN BA2B haplotype.

Differential production of the two most common allelic variants of beta-lactoglobulin (LG), beta-LG A and beta-LG B, has been observed using PAGE. This study evaluated 733 bp of the beta-LG promoter region and 92 bp of the first exon for possible polymorphisms using denaturing gradient gel electrophoresis and nucleotide sequence analysis. Within this region, 13 single nucleotide substitution polymorphisms were detected. Twelve polymorphisms were allele specific, and one appeared to be polymorphic only for the B allele. Several potential binding sites for transcription factors were found within the promoter sequence. This study investigated the role of the G to C transversion within a consensus binding site for activator protein-2 at position-430 bp upstream from the transcription initiation site. Using the DNase-I footprint assay, we confirmed the functional importance of this point mutation and showed different binding affinities of activator protein-2 for both alleles. We discuss the possible regulatory role of activator protein-2 in the transcriptional regulation of the beta-LG gene and propose the activator protein-2 transcription factor as a modulator of gene expression of beta-LG.

The high-growth (hg) locus in the mouse produces a 40% increase in adult body weight and proportional hypertrophy of skeletal muscles, due to fiber hyperplasia. Using biochemical and histological criteria we tested the hypothesis that myogenesis is delayed in fetal in high-growth mice, compared with normal mice, allowing an increase in the muscle stem cell population. Significant biochemical and weight differences between lines were first apparent at embryonic day 17 (E17). At this stage, high-growth hind limbs were smaller, contained fewer nuclei, less RNA, and showed less creatine kinase (CK) activity, than controls. Histologically, high-growth muscles contained fewer, but larger, myotubes and increased extracellular matrix (at E17) compared with controls. By embryonic day 19, high-growth limbs showed increases in wet weight, CK activity, protein, RNA, and DNA compared with controls. Our results are consistent with delayed production and/or fusion of high-growth myoblasts to form secondary myotubes from embryonic day 15 to 17 and accelerated production of secondary myotubes from day 17 to 19. Delayed fusion of high-growth myoblasts may allow an increase in the muscle stem cell population, resulting in fiber hyperplasia at birth.

The high growth (hg) locus in mice produces a 30-50% increase in weight gain of homozygous individuals. Here we report that the microsatellite marker D10Mit69 is deleted in high growth mice. The deletion of D10Mit69 was uncovered in a screen of the high growth mouse and its progenitor strains for available markers in the hg region. We demonstrate that hg and D10Mit69 cosegregate in a cross of congenic strains C57BL/6J-hghg x C57BL/6J. These results suggest that deletion of a region around D10Mit69 is responsible for the high growth effect. Marker D10Mit69 will be utilized as an entry point to physical cloning of the hg-containing segment. A dense map of markers around hg constructed here should allow identification of markers in homologous regions in domestic animals and humans, which may be utilized to assess the role of the hg locus in these other species.

The random amplified polymorphic DNA (RAPD) assay utilizes the polymerase chain reaction (PCR) and short primers of arbitrary nucleotide sequence to amplify DNA. In this study, the RAPD assay was used to identify and map polymorphic markers in the AgResearch International Mapping Flock (IMF) sheep pedigrees. Sires and dams of eight of the full-sib IMF pedigrees were screened with 131 different 10-mer oligonucleotide primers. An average of 85 RAPD polymorphisms was identified between each parental pair, and 53 markers were contributed to the AgResearch IMF collaboration. Forty-five of the RAPD markers were mapped in the AgResearch IMF genetic linkage map, and at least one marker was located on 17 of the 26 autosomes and both sex chromosomes. Three lines of evidence were used to check for the homology of scored polymorphisms in different pedigrees, pedigree evaluation, segregation analysis, and Southern blot analysis. These results demonstrate that the RAPD assay is a powerful approach for identifying polymorphisms that can be used as markers for constructing a sheep genetic linkage map.

Transgenic mice were produced by microinjection of a DNA construct composed of the bovine kappa-casein (kappa-CN) cDNA under the control of the goat beta-CN 5' promoter elements and 3' flanking regions into pronuclear-stage embryos. The gene construct targeted the expression of bovine kappa-CN RNA to the mammary gland and secretion of bovine kappa-CN in the milk. In the three lines studied (BC-7, BC-31 and BC-67) the transgene was stably integrated and propagated as a Mendelian locus. Expression of the bovine protein in lactating mice from the three transgenic lines was demonstrated by northern and western blots. In ten different tissues analysed by northern blotting, expression was confined to the mammary gland of lactating transgenic mice from line BC-7, with low-level expression also observed in the salivary gland of lines BC-31 and BC-67. Transgene expression in the mammary gland paralleled normal casein gene expression during lactation and was not observed in virgin females. The level of bovine kappa-CN mRNA expression on day 10 of lactation in hemizygous transgenic females in relation to endogenous mRNA of whey acid protein (WAP) gene expression was 14%, 69%, and 127% in lines BC-7, BC-31 and BC-67, respectively. No association between transgene copy number and expression was observed. The bovine kappa-CN concentration in milk on day 10 of lactation ranged from 0.94 to 3.85 mg of protein per ml of milk. The bovine kappa-CN expressed in mouse milk had the same molecular mass and immunoactivity with polyclonal antibodies as did kappa-CN from bovine milk. A high degree of variation in the production of bovine kappa-CN within each of the transgenic lines was observed.

kappa-Casein is the protein fraction of milk that allows formation of micelles and determines micelle size and function, thus affecting many of the physical characteristics of milk. Several lines of transgenic mice were generated bearing the B allele of the bovine kappa-CN gene under the control of the regulatory sequences of the caprine beta-CN gene that specifically directed expression of bovine kappa-CN to the lactating mammary tissue of these mice. High expression of bovine kappa-CN protein was observed in the lines studied; the total level of protein in milk was not significantly affected. A high degree of conservation in the amino acids involved in the predicted three-dimensional structure exists between murine and bovine kappa-CN. Milk from transgenic lines expressing high bovine kappa-CN had a significantly smaller micelle size than did control milk. Therefore, bovine kappa-CN appears to have effectively participated in assembly of murine casein micelles. There was no effect on the time of rennet coagulation, but the association was significant between the milk of transgenic lines and the production of a stronger curd in rennet-induced gels. We conclude that bovine kappa-CN is an appropriate candidate for transgenic technology that would increase the ratio of kappa-CN to the calcium-sensitive caseins, therefore affecting the physical properties of the colloidal casein suspension.

The importance of transcriptional control of gene expression has been documented in relation to several genetic defects in man. Mutations in the promoter region of genes have been shown to be responsible for defects which are characterized by over or under expression of genes. Genetic polymorphisms of the milk protein genes, and their high level of tissue specific expression during lactation, represent a suitable system for studying regulation of gene expression. Quantification of the allele specific protein products has demonstrated unequal allelic contribution in the expression of the beta lactoglobulin (beta-LG) alleles A and B. The DNA sequence analysis of the beta-LG promoter revealed allele specific polymorphisms related to the coding region genotypes.

High birth weights were observed in calves that developed from bovine embryos produced by in vitro maturation (IVM) and in vitro fertilization (IVF) procedures. After IVM and IVF, embryos were either co-cultured in vitro with oviductal epithelial cells or transferred into the sheep oviduct for development to the blastocyst stage. Blastocysts were transferred to the reproductive tracts of recipient heifers and cows for development to term. Birth weights and gestation periods were compared between calves that developed from in vitro-derived embryos and calves born after artificial insemination (AI) of cows in the herd from which recipient females were selected. Gestation periods were not different among the groups (P > 0.05), but calves that developed from IVM/IVF-derived embryos co-cultured in vitro were larger at birth than calves born from IVM/IVF-derived embryos that developed into blastocysts in the sheep oviduct and calves born from AI (P < 0.001). Dystocia and calf mortality were associated with large calf size at birth. These data were collected from an experiment designed for other purposes, and confounding variables and small sample size could have influenced the observed differences in birth weights. Nevertheless, the extreme birth weights of some calves suggest that abnormal prenatal growth occurs in some IVM/IVF-derived bovine embryos and that conditions for co-culture to the blastocyst stage may exacerbate the problem.

The objective of this work was to estimate the contribution of milk protein genotype to production traits of Holstein cattle. Two approaches were employed. The first was based on an animal model analysis of milk records in which cows were genotyped for milk protein variants. The second approach was the analysis of PTA of sires in which the sires were genotyped for their milk protein variants. Results of the animal model analysis agreed qualitatively with previously published analyses of cow records, indicating only a minor contribution of milk protein genotype to production traits. Qualitative similarity was also found between the analysis of sire transmitting abilities and the animal model. Algebraic results suggested, however, that an indirect analysis of published sire transmitting abilities does not provide unbiased estimates of differences in milk protein genotypes. Although analysis of PTA is simple, only a direct analysis of genotyped cows with an animal model provides unbiased estimates of genotype differences.

The high growth phenotype in the mouse results from a single autosomal recessive locus (hg) mapped to chromosome 10, that produces a marked increase in growth rate and body size compared to controls. Using morphometric methods, we quantified skeletal muscle growth in newborn male and female high growth mice, and at 3, 6, and 9 weeks of age. Adult male high growth mouse body weights increased by 42% and muscle weights increased 39-43% compared to controls. Differences between high growth and control muscle weights were removed after normalization to body weight. Similar changes in muscle and body weights were found in female high growth mice. Muscle cross-sectional area was significantly (P < 0.05) increased in high growth mouse gastrocnemius (GAS), soleus (SOL), and tibialis anterior (TA) muscles at 6 and 9 weeks of age. Cross-sectional areas of slow type 1, and fast type 2b fibers were moderately increased in all high growth muscles measured. Muscle fiber number was increased by approx 30% in the high growth SOL, GAS and TA muscles. We conclude that rapid growth in the high growth mouse is accompanied by an increase in muscle mass, due to fiber hyperplasia and hypertrophy, and that the hg locus may act during myogenesis to alter the proliferation rate and/or fusion kinetics of high growth mouse muscle cells.

Genotypes of 112 Israeli Holstein sires were determined for beta-lactoglobulin and kappa-casein by restriction fragment length polymorphism analysis of amplified DNA. Frequencies of the A alleles were .89 and .52 for kappa-casein and beta-lactoglobulin, respectively. Genotype frequencies were in accordance with Hardy-Weinberg equilibrium. Two models were used to test the effect of these loci on production of milk, fat, and protein and on fat and protein percentages. The dependent variable in the first model was production of 84,593 daughters of 110 genotyped sires, and the independent variables were herd-year-season, sire genotype for beta-lactoglobulin and kappa-casein, the interaction of the two loci, and sire nested within genotype. In the second model, the dependent variable was individual animal model genetic evaluations of 111 sires for production traits, and milk protein genotypes were the independent variables. The only significant effect of genotype with both models was the interaction of the two loci on fat percentage. When sires, rather than cows, were genotyped, the contrasted groups differed by only one-half of the genotype effect, but the residual variance was reduced.

This study was designed to measure the survival and development of pronuclear stage bovine zygotes after microinjection with DNA and either culture in vitro or incubation in the sheep oviduct and to determine the percentage of embryos that retain exogenous DNA at the blastocyst stage. In vitro matured and fertilized oocytes were examined for pronuclear development 18 to 20 h after coincubation with sperm. An ovine somatotropin gene construct was microinjected into ova with visible pronuclei. Microinjected ova and unmanipulated ova from the same in vitro derived pool were cocultured with oviductal epithelial cells or incubated in the sheep oviduct and cocultured, respectively, for 7 d. Blastocysts were subjected to polymerase chain reaction analysis for detection of exogenous DNA. The percentage of cleaved embryos that formed blastocysts was similar for microinjected and unmanipulated cultured ova. The percentage of blastocysts recovered from the sheep oviduct that hatched was higher than for blastocysts that developed in coculture. Fourteen of 26 (54%) blastocysts that developed from microinjected ova were positive for the exogenous DNA by polymerase chain reaction. The implications are discussed for polymerase chain reaction detection of the exogenous gene construct in a relatively high proportion of microinjected blastocysts.

We present a polymerase chain reaction (PCR)-based procedure for rapid bovine embryo sexing and classifying embryos for the presence of exogenous DNA. Fourteen bovine blastocysts microinjected with gene construct DNA at the pronuclear stage were divided into quarters and subjected to amplification with construct-specific and sex gene-specific (ZFY/ZFX) primers in the same initial PCR reaction. Blastocysts carrying microinjected construct DNA could be identified by the presence of construct-specific PCR product in approximately 4 h. Approximately half of the microinjected and two of 16 non-microinjected blastocysts typed PCR-positive for the construct DNA. Owing to erroneous amplifications in the two non-microinjected control blastocysts, and the inability of the system to distinguish integrated from non-integrated copies of the microinjected construct, the number of construct-positive blastocysts determined in our assay most likely overestimates the number of true transgenic embryos. Nevertheless, using this assay, we were able to determine that approximately half of the microinjected embryos were negative for the transgene construct and thus could be eliminated from transfer to a recipient cow. Embryo sexing was achieved in less than 6 h by restriction fragment length polymorphism analysis of nested ZFY/ZFX PCR products reamplified from initial PCR reactions. In 11/14 microinjected blastocysts all sections assayed unambiguously as the same sex. In one embryo, only one section was analysed, while two other blastocysts showed some discrepancies of sexing results between the sections analysed.(ABSTRACT TRUNCATED AT 250 WORDS)

Whole blood is a common source of DNA, especially for genotype diagnostic services. Many laboratories now receive blood samples by mail and/or store blood samples prior to DNA extraction. Therefore, the effects of storage time (number of days from blood collection to DNA extraction) and temperature on DNA yield and quality are important. We compared DNA yield and quality from bovine blood samples exposed to one of sixteen treatment combinations of storage time (3, 7, 14 or 28 days) and temperature (-20 degrees, 4 degrees, 23 degrees or 37 degrees C) to the results obtained when DNA was extracted within 4 h of collection. The highest mean DNA yields, relative to the control samples, were obtained from blood stored at 4 degrees C. Blood stored at 37 degrees C for > or = 3 days or 23 degrees C for > or = 7 days yielded less (P < .05) DNA, compared with blood stored at 4 degrees or -20 degrees C for up to 28 days. DNA was not recovered from three of the samples stored at 23 degrees C and five of the samples stored at 37 degrees C. The genomic DNA obtained was of high molecular weight and suitable for restriction enzyme digestion and PCR amplification, regardless of the treatment.

beta-Casein genetic variants A1, A2, and B were separated using cation-exchange fast protein liquid chromatography. beta-Casein from a herd bulk casein sample eluted as a series of three peaks. Casein samples from individual cows containing known combinations of beta-casein A1, A2, and B were used to confirm that the three peaks were beta-casein genetic variants. An acid-PAGE gel confirmed the identity of the peaks that eluted from the column.

We describe a quick and efficient method of determining the sex of DNA samples from humans, cattle, sheep, and goats. Using universal primers we have amplified 447 or 445 bp fragments from male or female genomic DNA corresponding to the ZFY or ZFX genes. Restriction fragment length polymorphism (RFLP) analysis of the fragments yielded specific banding patterns between the two sexes in these species. Horse, pig and rainbow trout fragments did not show RFLPs with the group of enzymes chosen for the other species. Use of this method would allow animal breeders to determine the sex of preimplantation embryos before transfer to recipient females. Human embryos at risk for sex-linked disorders could be sexed soon after in vitro fertilization (IVF) and options considered before implantation.

The milk protein genotype of a mammary gland tissue that was used to generate a cDNA library at the University of California, Davis was typed by PAGE. Casein and whey proteins were extracted from the mammary tissue and typed utilizing fast mini-gel procedures that provide excellent resolution of the milk proteins. The genotype of the mammary tissue was classified as kappa-casein AB, beta-casein A2A1, alpha s1-casein BB, beta-lactoglobulin AA, and alpha-lactalbumin BB. The genes kappa-casein A, beta-casein A2, beta-lactoglobulin A, and alpha-lactalbumin B that have been cloned from this cDNA library coincide with those in the above tissue genotype with the exception of the recently reported cloning of the alpha s1-casein A gene. The gene frequencies for the casein genetic variants for three breeds of dairy cattle is presented and discussed in relation to the low frequency (.3%) of the alpha s1-casein A in the population.

Growth rate, body composition, cell number, cell size, and the activity of four dehydrogenase enzymes were studied from 10 to 25 days of age in one control (1C) and three lines (3, 9, 10) of Tribolium castaneum that had been subjected to long term selection for large 21-day pupae weight.-Selected lines were two- to three-fold larger in size than the control line throughout development. No major differences in percent of protein were detected among the lines but at any particular age, the selected lines were found to have a higher fat content than the control line. The differences in fat content were closely correlated with development such that all the lines reached very similar levels of percent of fat just prior to pupation. Water content showed an inverse relation to percent of fat.-Selection was observed to have caused major changes in the cellular response to growth. The selected lines had an average of from 17% to 48% larger cells (measured as protein/DNA) and were found to have from 37 to 62% more cells (measured as total DNA) than the control line at all ages from 10 to 19 days of age. In addition, the selected lines had a higher RNA content at all ages studied and higher RNA:DNA ratios at the young ages. In contrast the enzyme activities of ICDH and LDH were 60% lower. The results are interpreted as indicating that a more efficient metabolic machinery had evolved in the rapidly growing selected lines.

Food consumption, feed efficiency, metabolic rate and glucose utilization were studied throughout development in one control (1C) and three selected lines (3, 9, 10) of Tribolium castaneum that had been subjected to long term selection for 21-day pupae weight. Growth rate, body composition, cellular growth and the activity of four dehydrogenase enzymes in the same lines have been reported (Medrano and Gall 1976).-Larva of selected lines consumed 1.2 times as much food as the control and gained an average of 2.9 times as much weight. The rapid growth of the selected lines was associated with a gross feed efficiency 20 to 30% above that for the control line. There was also a small but consistent improvement in the conversion of digested food. Average digestibility was higher for selected lines.-There was little apparent differentiation between the control and selected lines in metabolic rate/individual, but the rate measured on a per-unit weight basis was two- to three-fold greater for the control during the active growth stages. Respiratory quotients (R.Q.) of 1.0, indicative of carbohydrate oxidation, were observed through larval growth in all lines. Pupae at 21 days showed R.Q. values greater than 1.0, which were interpreted as resulting from a phenomenon in insects in which CO(2) is released by pupae, in large bursts at irregular intervals. The rate constant of glucose oxidation, measured as the rate of C(14) labelled CO(2) respired during 2- to 6-hour incubation periods, was two- to three-fold higher in the control. In addition, the control line larvae expired 5% to 17% more of the ingested C(14) as CO(2). It was apparent that control line individuals maintained a much more active turnover of metabolites but without an effective retention of carbon as body substances. The results are discussed in support of the hypothesis that selection for large body size resulted in improved control mechanisms that influence the biological efficiency of growth in Tribolium.