Linkage relationship among loci determining genetic variants of serum and milk proteins in cattle

Special methods allowing usage of inadequate pedigrees were employed to examine linkage among the milk protein loci alpha s1-casein, beta-casein, chi-casein and beta-lactoglobulin, and the loci for serum amylase, ceruloplasmin and transferrin. Linkage was evident between the alpha s1-casein and beta-casein loci, the alpha s1 and chi-casein loci, the beta-casein and chi-casein loci, also amylase and transferrin loci. Recombination fractions for these corresponding combinations were 0.00; 0.00; 0.00 and 0.30. Weak linkage (recombination fraction being 0.46; 0.44 and 0.42) between the beta-lactoglobulin and beta-casein loci, the amylase and ceruloplasmin loci, ceruloplasmin and transferrin loci is supposed.     

New genetic variants identified in donkey's milk whey proteins

Novel genetic variants for donkey milk lysozyme and -lactoglobulins I and II have been identified by the combined use of peptide mass mapping and sequencing by tandem mass spectrometry in association with database searching. The novel donkey lysozyme variant designated as lysozyme B (Mr 14,631 Da) differed in three amino acid exchanges, N⁴⁹ D, Y⁵² S, and S⁶¹ N, from the previously published sequence. Three novel genetic variants for donkey -lactoglobulins were identified. One of them is a type -lactoglobulin I with three amino acid exchanges at E³⁶ S, S⁹⁷ T, and V¹⁵⁰ I (-lactoglobulin I B, Mr 18,510 Da). The two others are type -lactoglobulins II with two amino acid exchanges at C¹¹⁰ P and M¹¹⁸ T (-lactoglobulin II B, Mr 18,227 Da) and with three amino acid exchanges at D⁹⁶ E, C¹¹⁰ P, and M¹¹⁸ T (-lactoglobulin II C, Mr 18,241 Da). All these primary structures are closely related to those of homologous proteins in horse milk (percent identity >96%).     

Quantitative differences between variants of A spermatogonia in man

The area of cytoplasm, nucleus, nucleolus and mitochondria, as well as the elongation and irregular outline of the nucleus were determined, on electron micrographs by using an image analyser, for Ap (pale), Ad (dark with intranuclear vacuole), Ad-like (dark without intranuclear vacuole), Ac (cloudy) and Al (long) human spermatogonia. Ap and Ac spermatogonia had larger nucleus, larger nucleolus, and more cytoplasm than did Ad, Ad-like, and Al spermatogonia. In addition, the nuclei of Ap and Ac spermatogonia were more spherical and had a more distinct outline.     

Haemoglobin variants in cattle

From a comparative survey of reports on cattle Hb variants it is concluded that prior to the present communication the occurrence of at least seven different adult Hb molecules have been reported.
In addition comes the finding of a new Hb variant called HbG which is reported in this study. This was found in three animals among 101 East African Zebu cattle. It migrated more slowly than any cattle Hb variant previously reported and made up 20% of total Hb.
The gene frequencies of the East African Zebu population were: Hb^A = 0.52, Hb^B = 0.32, Hb^c = 0.14 and Hb^G = 0.01.     

Identification of allele variants of cattle milk productivity genes using PCR anti-primer method

In this work we have demonstrated two independent real-time PCR methods for detection single nucleotide polymorphism (SNP) of genes csn and acyl-CoA: diacylglycerol acyltransferase 1 (dgat) in cattle. We have analyzed 296 samples of milk production cattle of Ukrainian breeding. The genotype frequencies were AA--0.58, AB--0.34, BB--0.08 for csn gene and for dgat gene--AA--0.7, AK--0.26, KK--0.04. High efficiency of so called "anti-primer" method was shown. Duration of anti-primer PCR reaction was about 2-2.5 hours only and provided full investigation of unknown gene allele.     

Tertiary and quaternary structural differences between two genetic variants of bovine casein by small-angle X-ray scattering

The casein complexes of bovine milk consist of four major protein fractions, alpha s1, alpha s2, beta, and kappa. Colloidal particles of casein (termed micelles) contain inorganic calcium and phosphate; they are very roughly spherical with an average radius of 650 A. Removal of Ca2+ leads to the formation of smaller protein aggregates (submicelles) with an average radius of 94 A. Two genetic variants, A and B, of the predominant fraction, alpha s1-casein, result in milks with markedly different physical properties, such as solubility and heat stability. To investigate the molecular basis for these differences, small-angle X-ray scattering was performed on the respective colloidal micelles and submicelles. Scattering curves for submicelles of both variants showed multiple Gaussian character; data for the B variant were previously interpreted in terms of two concentric regions of different electron density, i.e., a "compact" core and a relatively "loose" shell. For the submicelle of A, there was a third Gaussian, reflecting a negative contribution due to interparticle interference. Molecular parameters for submicelles of both A and B are in agreement with hydrodynamic data in the literature. Data for the micelles, for which scattering yields cross-sectional information, were fitted by a sum of three Gaussians for both variants; for these, the corresponding two lower radii of gyration represent the two concentric regions of the submicelles, while the third reflects the average packing of submicelles within the micellar cross section. Most of the molecular parameters obtained showed small but consistent differences between A and B, but for submicelles within the micelle several differences were particularly notable: A has a greater molecular weight for the "compact" region of the constituent submicelle (82,000 vs 60,000) and a much greater submicellar packing number (6:1 vs 3:1). Reasons for these and other differences are to be sought in sequence differences and in differences in calcium-binding sites and charge distribution.     

Expected early genetic gain from selection for milk yield in dairy cattle

Summary A matrix program to predict short term genetic gain from single trait selection for milk yield was developed. Rate of genetic gain was calculated as the annual change in the mean breeding value of all producing females. Several parameters sets representing various selection policies were used to examine situations pertinent to dairy populations of the United States. Approach to the asymptotic rates of genetic gain within the model varied with the choice of parameters, but even with consistent selection policies, predicted total genetic gain in the first 10 years was only half of the expected from classical theory. Considerable year to year variation in the rate of gain occurred. Early gains were more dependent on female selection decisions than gains during the steady state. In a two-phase model, the approach to the linear rate of gain in the second phase was accelerated by starting with an ongoing improvement program, but considerable delays still existed. Selection for sex- limited traits such as milk yield, which require pedigree selection and a waiting time for progeny test results reached asymptotic rates more slowly than previously assumed.     

Procedure for simultaneous phenotyping of genetic variants in cow's milk by isoelectric focusing

A method is described which allows the simultaneous separation of all polymorphic protein fractions in cow's milk in one single run. The separation could be achieved by isoelectric focusing in ultrathin-layer polyacrylamide gels. The method is especially suitable for screening purposes because it combines low costs, high resolution and short separation time.     

Functional differences between TRPC4 splice variants.

Functional characterizations of heterologously expressed TRPC4 have revealed diverse regulatory mechanisms and permeation properties. We aimed to clarify whether these differences result from different species and splice variants used for heterologous expression. Like the murine beta splice variant, rat and human TRPC4beta both formed receptor-regulated cation channels when expressed in HEK293 cells. In contrast, human TRPC4alpha was poorly activated by stimulation of an H(1) histamine receptor. This was not due to reduced expression or plasma membrane targeting, because fluorescent TRPC4alpha fusion proteins were correctly inserted in the plasma membrane. Furthermore, currents through both human TRPC4alpha and TRPC4beta had similar current-voltage relationships and single channel conductances. To analyze the assembly of transient receptor potential channel subunits in functional pore complexes in living cells, a fluorescence resonance energy transfer (FRET) approach was used. TRPC4alpha and TRPC4beta homomultimers exhibited robust FRET signals. Furthermore, coexpressed TRPC4alpha and TRPC4beta subunits formed heteromultimers exhibiting comparable FRET signals. To promote variable heteromultimer assemblies, TRPC4alpha/TRPC4beta were coexpressed at different molar ratios. TRPC4beta was inhibited in the presence of TRPC4alpha with a cooperativity higher than 2, indicating a dominant negative effect of TRPC4alpha subunits in heteromultimeric TRPC4 channel complexes. Finally, C-terminal truncation of human TRPC4alpha fully restored the channel activity. Thus, TRPC4beta subunits form a receptor-dependently regulated homomultimeric channel across various species, whereas TRPC4alpha contains a C-terminal autoinhibitory domain that may require additional regulatory mechanisms.     

Procedure for simultaneous phenotyping of genetic variants in cow's milk by isoelectric focusing*

A method is described which allows the simultaneous separation of all polymorphic protein fractions in cow's milk in one single run. The separation could be achieved by isoelectric focusing in ultrathin-layer polyacrylamide gels. The method is especially suitable for screening purposes because it combines low costs, high resolution and short separation time.     

Identification of allele variants of cattle milk productivity genes using PCR and the anti-primer method

In this study, we present two independent approaches in using Real-Time PCR for the detection of single nucleotide polymorphism (SNP) in genes encoding a kappa-casein and an acyl CoA: diacylglycerol acyltransferase 1 (DGAT 1) in cattle. The samples from 296 Ukrainian-bred dairy cows were analyzed. The genotype frequencies for the kappa-casein gene were as follows: AA, 0.58; AB, 0.34; and BB, 0.08; the frequencies for the acyl-CoA: diacylglycerol acyltransferase 1 gene were as follows: AA, 0.7; AK, 0.26; and KK, 0.04. The high efficiency of the anti-primer method was shown. The anti-primer Real-Time PCR test takes only 2–2.5 h and allows for the complete identification of the unknown allelic variant of the studied genes.     

Database of cattle candidate genes and genetic markers for milk production and mastitis

A cattle database of candidate genes and genetic markers for milk production and mastitis has been developed to provide an integrated research tool incorporating different types of information supporting a genomic approach to study lactation, udder development and health. The database contains 943 genes and genetic markers involved in mammary gland development and function, representing candidates for further functional studies. The candidate loci were drawn on a genetic map to reveal positional overlaps. For identification of candidate loci, data from seven different research approaches were exploited: (i) gene knockouts or transgenes in mice that result in specific phenotypes associated with mammary gland (143 loci); (ii) cattle QTL for milk production (344) and mastitis related traits (71); (iii) loci with sequence variations that show specific allele-phenotype interactions associated with milk production (24) or mastitis (10) in cattle; (iv) genes with expression profiles associated with milk production (207) or mastitis (107) in cattle or mouse; (v) cattle milk protein genes that exist in different genetic variants (9); (vi) miRNAs expressed in bovine mammary gland (32) and (vii) epigenetically regulated cattle genes associated with mammary gland function (1). Fourty-four genes found by multiple independent analyses were suggested as the most promising candidates and were further in silico analysed for expression levels in lactating mammary gland, genetic variability and top biological functions in functional networks. A miRNA target search for mammary gland expressed miRNAs identified 359 putative binding sites in 3'UTRs of candidate genes.     

Genetic differences in milk proteins among laboratory rats (Rattus norvegicus)

Genetic differences in the electrophoretic pattern of milk proteins were observed among different inbred rat strains. The results of segregation analysis made on a limited number of females is consistent with the hypothesis of two codominant autosomal alleles system.     

Genome-wide associations for investigating time-dependent genetic effects for milk production traits in dairy cattle

Phenotypic variation in milk production traits has been described over the course of a lactation as well as between different parities. The objective of this study was to investigate whether variation in production is affected by different loci across lactations. A genome-wide association study (GWAS) using a 50-k SNP chip was conducted in 152 divergent German Holstein Friesian cows to test for association with milk production traits over different lactations. The first four lactations were analysed regarding milk yield, fat, protein, lactose, milk urea nitrogen yield and content as well as somatic cell score. Two approaches were used: (i) Wilmink curve parameters were used to assess the genetic effects over the course of a lactation and (ii) test-day yield deviations (YD) were used as a normative approach for a GWAS. The significant effects were largest for markers affecting curve parameters for which there was a statistical power <0.8 of detection even in this small design. While significant markers for YDs were detected in this study, the power to detect effects of a similar magnitude was only 0.11, suggesting that many loci may have been missed with this approach in the present design. Furthermore, all significant effects were specific for a single lactation, leading to the conclusion that the variance explained by a certain locus changes from lactation to lactation. We confirm the common evidence that most production traits vary in the degree of persistency after the peak as a result of genetic influence.     

Rapid selection of genetic and antigenic variants of foot-and-mouth disease virus during persistence in cattle.

Rapid evolution of foot-and-mouth disease virus (FMDV) is documented during persistent infections of cattle. The carrier state was established experimentally with plaque-purified FMDV of serotype C3. Virus was recovered from the esophageal pharyngeal area of the animals up to 539 days postinfection. Analysis of capsid proteins by electrofocusing and by electrophoretic mobility of the genomic poly(C)-rich tract suggested heterogeneity in several isolates and sequential dominance of viral subpopulations. Nucleotide sequences of the VP1-coding region of the parental FMDV C3 clones and of seven isolates from the carrier cattle showed point mutations that represented rates of fixation of mutations of 0.9 X 10(-2) to 7.4 X 10(-2) substitutions per nucleotide per year; 59% of the base changes led to amino acid substitutions, some of which were located within residues 135 to 151, a region involved in neutralization of FMDV. In the esophageal pharyngeal fluid samples, FMDV C3-neutralizing activity was present. Antigenic variation was demonstrated with monoclonal antibodies raised against FMDV C3. Two isolates from carrier cattle differed from the parental virus by 10(2)- or 10(3)-fold decreased reactivity with neutralizing monoclonal antibodies. We suggest that persistent, inapparent infections of ruminants, in addition to being a reservoir of virus, may promote the rapid selection of antigenically variant FMDVs.