The Mac-T cell system was used to investigate the role of the prolactin (PRL) receptor in cell proliferation and the regulation of milk protein synthesis. This study was designed to investigate whether overexpressing the PRLR in the Mac-T cell line resulted in a change in its growth rate and an enhancement of its ability to produce milk proteins. To accomplish these goals, Mac-T cells were stably transfected with the rabbit prolactin receptor gene. Fifteen clones and a pool of transfectants were obtained. Of these, one clone and the pool were positive for the PRL receptor expression. The clone (S15) and pool (SP) cells were sorted into high (H), medium (M), and low (L) expressors, of the PRLR. The high expressors were used for all subsequent experiments. The presence of high levels of the PRLR on the surface of S15 and SP cells was further confirmed by receptor binding assay and Western Blot. Following the establishment of these cell lines, the cells were used to investigate the effect of increased levels of PRLR on cell proliferation and milk protein synthesis. / It was found that the growth rate of parental cells was depressed in the presence of 5 mug/ml of PRL. In contrast, the growth rate of the transfectants was enhanced by the addition of 5 mug/ml PRL to the culture medium. In addition, both "SP" and "S15" cells produced higher levels of STAT5 upon long-term (48 h) PRL stimulation. No effect on the synthesis of alpha S1- and beta-caseins was noted. It is likely that no differences in protein synthesis were observed because the cells have lost the ability to differentiate, even when cultured on collagen gels in the presence of lactogenic hormones.

Methodologies have been devised to genotype the $ kappa$-casein ($ kappa$-CN) and $ beta$-lactoglobulin ($ beta$-LG) loci using the polymerase chain reaction (PCR) and restriction fragment length polymorphisms (RFLPs) analysis. A fragment of 432 base pair from the $ kappa$-CN gene which contains the nucleotide substitutions diagnostic of the A- and B-alleles of $ kappa$-CN was amplified. DNA amplified from the A-variant contained a unique HinfI restriction site, whereas DNA amplified from the B-variant contained a unique TaqI site. Thus, the genotype of the animal could be determined by restriction enzyme analysis. A region extending from exon IV to V of the $ beta$-LG gene was also amplified using PCR. In this region, the B-allele contains 2 HaeIII restriction sites which are not present in DNA amplified from the A-allele. The digestion of the PCR product with HaeIII thus allowed discrimination between the A- and B-alleles. Analysis of bulls (n = 68) used by artificial insemination (AI) centres after 1980, bulls (n = 27) used before 1960, a random sample of male calves (n = 102) and cows (n = 123) revealed a frequency of the B allele of $ kappa$-CN as 0.13, 0.37, 0.22 and 0.19 in these populations, respectively. In the populations of bulls (n = 69) used by AI centres after 1980, bulls (n = 27) used before 1960, a random sample of male calves (n = 99), and cows (n = 129), the B-allele of $ beta$-LG was present at frequencies of 0.60, 0.52, 0.67, and 0.65, respectively. Statistical analysis of these data shows the frequency of the B-allele of $ kappa$-CN was significantly higher in sires (n = 27) which were used by the AI centre before 1960 compared to more modern proven sires (n = 68) (P $<$ 0.01).

Single-strand conformation polymorphism analysis (SSCP) and restriction fragment length polymorphism analysis (RFLP) were used to determine the genotype of Holsteins at the $ kappa$-casein ($ kappa$-CN) locus. A 432-bp fragment within exon IV containing nucleotide substitutions diagnostic of the A- and B-variants of $ kappa$-CN was amplified using the polymerase chain reaction (PCR). The sires from the earliest years of the AI industry had a significantly higher (p $<$ 0.01) frequency of allele than sires in modern usage. These data indicate that selection or milk production parameters may discriminate against the B-allele. SSCP analysis was also used for detecting polymorphisms within the regulatory region of $ kappa$-CN gene. A 640-bp fragment within the 5$ sp prime$-flanking region of bovine $ kappa$-CN gene which contained the TATA box, CAAT box, and exon I was amplified using PCR. The SSCP analysis of this fragment revealed no variation, possibly due to the lower detection efficiency of SSCP with large fragment size. Nested primers were, therefore, designed to amplify fragments of 234- and 486-bp. Polymorphism was detected only in the 486-bp fragment and the two variants were designated M$ sb1$ and M$ sb2.$ The allelic frequencies of M$ sb1$ and M$ sb2$ in bulls used by AI industry before 1970 were 0.67 and 0.33, and in bulls used by AI industry after 1980 the frequencies were 0.68 and 0.32, respectively. The frequency of these alleles were not significantly different in Holsteins used by AI industry before 1970 and after 1980. Unlike the apparent change in frequency of the A- and B-variants noted within exon IV, this polymorphism seems to have not responded to selection. However, a higher frequency of M$ sb1$ allele appeared to be associated with B-variant (exon IV) genotypes. The presence of these variants within the regulatory region may possibly be involved in the quantitative expression of $ kappa$-CN gene. (Abstract shortened by UMI.)

A genetic approach to lowering protease (plasmin) levels in milk, requires the presence of polymorphism of bovine plasminogen. This study was conducted to determine to what extent genetic polymorphism exists in dairy cattle. Bovine plasminogen was first purified from Holstein cow plasma by affinity chromatography on Lysine-Sepharose and antibodies to bovine plasminogen were raised by monthly intramuscular injection of the isolated bovine plasminogen into rabbits. For plasminogen phenotyping, blood samples were collected at random from 50 Holstein and Ayrshire cattle, and plasminogen was isolated from the plasma using lysine-Sepharose and then treated with neuraminidase. After separation by isoelectric focusing (pH 3.5-9.5) in polyacrylamide gels, Plasminogen polymorphs were detected immunologically using rabbit anti-bovine plasminogen antibodies. Additionally, the plasminogen isoforms were evaluated with a functional assay (caseinolytic overlay technique) after activation of the plasminogen with urokinase. Six plasminogen phenotypes were identified which represent products of 5 variant alleles. The 5 plasminogen variants were characterized based on their isoelectric points and designated PLG A$ sb2$ (pI 6.5 and 7.0), B$ sb2$ (pI 7.6 and 7.8), C$ sb1$ (pI 6.8), D$ sb2$ (pI 7.8 and 8.0), and E$ sb2$ (pI 6.8 and 7.0). PLG A$ sb2$ and PLG B$ sb2$ were the most common variants in these cattle. The 6 phenotypes were $ rm A sb2A sb2, B sb2B sb2, A sb2B sb2, B sb2C sb1, A sb2D sb2 and D sb2E sb2$. The phenotypic frequencies in Holstein and Ayrshire were very different, $ rm A sb2A sb2 and B sb2B sb2$ being respectively the most frequent phenotype. In addition, DNA polymorphism at bovine plasminogen gene was detected when genomic DNA was digested with the restriction enzyme Msp I and hybridized with mouse plasminogen cDNA. This is the first description of plasminogen polymorphism reported in dairy cattle. If different variants have altered activity, the detrimental effect

Includes bibliograpical references (leaves 119-134). The most common chromosome abnormalities in live cattle are various Robertsonian translocations (centric fusions). Two hypotheses have been used to explain how monocentric Robersonian translocation chromosomes are generated: either direct formation, or evolution from dicentic chromosomes. Four main cattle procentric Satellite sequences were used as single and two-colour fluorescence in situ hybridization probes for studying the centromere organisation of cattle autosomes and the rearrangement in two cattle Robertsonian translocation chromosomes, the t(1:29) which is monocentric and found in numerous breeds, and the t(14:20) which is dicentric and found in 2 breeds.

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Thesis (MScAgric )--University of Stellenbosch, 2001. / ENGLISH ABSTRACT: The main object of this study was to estimate heritabilities and possible genetic correlations for and between reproductive traits in a composite multi breed beef cattle herd. Reproduction is a complex process with many components. Due to the nature of the data, obtained from the two farms of the Johannesburg Metropolitan Council from 1974 to 1993, only calving date (CD), calving date with a penalty score (CDP), calving success (CS), calving interval (CI), age at first calving (AFC), longevity and stayability at 36, 48, 60, 72 and 84 months were investigated. A GFCAT set of programmes was used and fitted on a sire model to analyse all the categorical traits. Heritabilities and product moment correlations between predicted breeding values for stayability at 36, 48, 60, 72, 84 months, calving success (CS) and longevity, were estimated. The estimated heritabilities on the underlying scale for these traits were 0.06, 0.10, 0.06, 0.03, 0.11, 0.03 and 0.08, respectively. Product moment correlations between breeding values for stayability traits were very low. The highest correlation of 0.22 was obtained between 36 and 48 months. Heritability estimates and correlations between traits appear to be of such a low magnitude that selection for these characteristics would result in limited improvement and indicate that the sire had little influence on his daughter's stayability, longevity and CS. For the analyses oflinear traits (CI, CD, CDP and AFC), a REML procedure fitting a multitrait animal model (using REML VCE 4.2.5 package of Groeneveld, 1998) was used. Heritabilities and geneticcorrelations for and between calving interval (CI), calving date (CD), calving date with a penalty score (CDP) and age at first calving (AFC) were estimated as traits of the dam. The estimated heritabilities for CI, CD, CDP and AFC obtained in this study were 0.01, 0.04, 0.06 and 0.40, respectively with a repeatability of 0.07, 0.12 and 0.13 for CI, CD and CDP, respectively. Genetic correlations between traits obtained varied from low to moderate, except for the high correlations between CD and CDP (0.98), CI and CD (0.75) and CI and CDP (0.79). Heritabilities, genetic correlations and repeatabilities of CD and CDP obtained in this study suggest that CD and CDP are the same traits and that selection for CDP rather than for CD does not have any additional advantage. Due to the additional advantages of CD over CI and the fact that CD is a less biased measurement of the female reproductive complex, CD appears to be of genetic value and should.be considered-as a possible selection criterion to ensure genetic improvement for reproduction in a beef cattle herd. / AFRIKAANSE OPSOMMING: Die doeI met hierdie studie was om oorerflikhede vir en moontlike genetiese korrelasies tussen verskillende reproduksiekenmerke in 'n meerras vleisbeeskudde te bepaal. Reproduksie is 'n komplekse proses en bestaan uit veelvuldige komponente. Weens die aard van die data, verkry vanaf die Johannesburg Metropolitaanse Raad se twee plase (vanaf 1974 tot 1993), is slegs kalfdatum (CD), kalf datum met 'n verswaringswaarde (CDP), kalwingsukses (CS), kalfinterval (CI), ouderdom met eerste kalwing (AFC), langlewendheid en volhoubaarheid van die moeders om reproduktief in die kudde tot op onderskeidelik 36, 48, 60, 72 en 84 maande te bly, bestudeer. Kategoriese kenmerke is deur middel van 'n GFCAT stel programme, wat op 'n vadermodel gepas is, geanaliseer. Oorerflikhede vir en die produkmoment korrelasies tussen die voorspelde teelwaardes vir CS, langlewendheid en volhoubaarheid op onderskeidelik 36, 48, 60, 72 en 84 maande, is bereken. Die oorerflikhede vir bogenoemde kenmerke was onderskeidelik 0.03, 0.08, 0.06, 0.10, 0.06, 0.03 en 0.11. Die korrelasie tussen die voorspelde teelwaardes vir die verskillende volhoubaarheidskenmerke was laag. Die hoogste korrelasie, n1. 0.22, is tussen 36 en 84 maande verkry. Die oorerflikhede en korrelasies tussen die kenmerke blyk van so 'n lae omvang te wees dat direkte seleksie vir die kenmerke slegs tot 'n beperkte genetiese verbetering sal lei en dui daarop dat die vader slegs 'n beperkte invloed op CS, volhoubaarheid en die langlewendheid van sy dogters het. Vir die analise van die lineere kenmerke (CI, CD, CDP en AFC), is 'n REML-prosedure gebruik wat op 'n multikenmerk dieremodel gepas is (deur die gebruik van die REML VCE 4.2.5 pakket van Groeneveld, 1998). Oorerflikhede vir en genetiese korrelasies tussen CI, CD, CDP en AFC is bereken as kenmerke van die moeder. In hierdie studie is die beraamde oorerflikhede vir CI, CD, CDP en AFC as onderskeidelik 0.01, 0.04, 0.06 en 0.40 bepaal, met herhaalbaarhede van onderskeidelik 0.07, 0.l2 en 0.13 vir CI, CD en CDP. Genetiese korrelasies tussen die kenmerke het van laag tot matig gevarieer, behalwe vir die hoe korrelasies tussen CD en CDP (0.98), CI en CD (0.75) en CI en CDP (0.79). Die oorerflikhede en herhaalbaarhede vir en genetiese korrelasie tussen CI en CDP verkry in hierdie studie, veronderstel dat CD en CDP in wese dieselfde kenmerk is en dat seleksie vir CDP in plaas van CD geen addisionele voordele inhou nie. Weens die addisionele voordele wat CD inhou, bo die van CI, en die feit dat CD 'n minder sydige bepaling van die vroulike reproduksiekompleks is, blyk CD van genetiese waarde te wees en moet dit as 'n moontlike seleksie kriterium, om genetiese verbetering in 'n vleisbeeskudde te verseker, oorweeg word.