Optimization of DNA blot hybridization conditions for various types of membranes]

A set of experiments has been conducted to choose the optimal conditions for DNA transfer and fixation on two types of the nitrocellulose, three types of nylon membranes and on capron filters. The buffer capillary transfer systems, electroblotting and gel hybridization are analyzed. Two techniques for DNA binding have been tested under different transfer conditions for all the membrane types: a vacuum fixation at 80 degrees C and a UV-exposure. The results indicate the critical dependence of the efficiency of blot-hybridization on the conditions of UV-treatment. The UV-exposure longer or shorter than the optimal one resulted in a loss of the hybridization efficiency. The optimal DNA-transfer and fixation conditions are recommended for all the membranes tested. The dependence of the optimal transfer and binding conditions on the specific characteristics of different membrane types was demonstrated for maximal sensitivity of the blot-hybridization.     

Changes in the methylated sequences and molecular population of wheat DNA during germination

The fractions of unique (Cot less than 405), moderately (Cot=0.13--405) and highly reiterated (Cot less than 0--0.13) sequences were isolated from DNA of wheat seeds and 3 day old seedlings, and GC content, amount of 5-methylcytosine and its distribution among various pyrimidine isostichs in the fractions isolated were studied. Different in Cot value DNA fractions from seeds or from seedlings are similar in GC content and in all other characteristics studied. Seed DNA differs from DNA of seedlings in the content of pyrimidine isostichs from the respective fractions of reiterated sequences. Pronounced differences in the amount of pyridmidine clusters with various base composition in the corresponding fractions of DNA from seeds and seedlings were found. These differences in the frequencies of respective pyrimidine clusters from DNA of seeds and seedlings may be considered as being a result of changes in the molecular population of wheat DNA on germination. The seed and seedling DNA differ significantly in the 5-methylcytosine content in the respective pyrimidine isostichs isolated from unique sequences. In the seedling DNA some other nucleotide sequences are to be methylated as compared to DNA of dormat seeds. Thus, on germination some changes occur in DNA methylation as well as in the genome organization.     

Characterization of the Russian beef cattle breed gene pools using inter simple sequence repeat DNA analysis (ISSR analysis)

The gene pools of beef cattle breeds bred in Russia were characterized on the basis of inter simple sequence repeat DNA analysis (ISSR analysis). Samples of Aberdeen Angus, Kalmyk, and Kazakh Whitehead breeds from Russia, as well as of Hereford breed, hybrids of Kazakh Whitehead and Hereford breeds, and Kazakh Whitehead breed from the Republic of Kazakhstan, were examined. In the examined breeds, 27 AG-ISSR fragments were identified, 25 of which were polymorphic. The examined breeds were different both in the fragment profiles (the presence/absence of individual ISSR fragments) and in their frequencies. It was demonstrated that the hybrid animals lacked some ISSR fragments that were present with high frequencies in parental forms, suggesting considerable genome rearrangement in the hybrid animals (at the regions of microsatellite localization) in crossings of the individuals from different breeds. The level of genetic diversity in Russian beef breeds was consistent with the values typical of farmed populations (breeds). The genetic diversity parameters assessed by applying Nei’s gene diversity index and the Shannon index varied from 0.0218 to 0.0605 and from 0.0225 to 0.0819, respectively. The highest Shannon index value was detected in the Kalmyk breed (0.0837) and Kazakh Whitehead breed from Russia (0.0819), and the highest level of Nei’s gene diversity index was found in the Kalmyk breed (0.0562) and in both populations of the Kazakh Whitehead breed (0.0509 and 0.0605). The high level of genetic similarity (according to Nei) was revealed between Russian beef cattle breeds and Hereford cattle: 0.839 (for the Kazakh Whitehead breed from Russia) and 0.769 (for the Kalmyk breed).     

Polymorphism of cattle prolactin gene: microsatellites, PSR-RFLP]

In the samples of Russian Ayrshire and Gorbatov Red cattle breeds, distribution of frequencies of prolactin (PRL) gene alleles generated due to the presence of polymorphic RsaI site in exon 3 were studied. In the breeds, the frequencies of the B allele of the PRL gene (with RsaI(+) site) detected by the PCR-RFLP method were 14.1 and 8.6%, respectively. In Black Pied, Ayrshire and Gorbatov Red cattle breeds, variation of the microsatellite dinucleotide repeat in the regulatory region of the gene PRL was also studied. Gorbatov Red breed was monomorphic at the microsatellite locus with the only allele 164 bp in length. Two alleles (164 bp and 162 bp) were detected in the other breeds studied. The frequencies of 164-bp allele of the microsatellite locus were 93.7 and 90.0% in Black Pied and Ayrshire breeds, respectively. In Gorbatov Red breed of dairy type with good beef qualities and low milk-fat yield, lower level of heterozygosity for PRL gene was demonstrated compared to Ayrshire and Black Pied breeds with high milk-fat yield. In three cattle breeds, higher mean estimate of polymorphism information content of PCR-RFLP in exon 3 (PIC = 0.21) was revealed compared with the same estimate (PIC = 0.09) for the microsatellite locus variability in the regulatory region of the PRL gene. Characteristics of allele B distribution of the PRL gene in the representatives of the Bovidae family are considered.     

Distribution of <Emphasis Type="Italic">BoLA-DRB3</Emphasis> allelic frequencies and identification of a new allele in the Iranian cattle breed Sistani (<Emphasis Type="Italic">Bos indicus</Emphasis>)

The distribution of the frequencies of BoLA-DRB3 gene alleles in the Iranian cattle breed Sistani was studied by the PCR-RFLP (“hemi-nested”) assay using restriction endonucleases RsaI, HaeIII and BstYI. In the examined cattle breed (65 animals) 32 alleles have been identified one of which being described for the first time (6.15% frequency). The nucleotide sequence of the polymorphic region of exon 2 of this allele has been determined and submitted in the GenBank database under accession number DQ486519. The submitted sequence has maximum homology (92%) with the previously described sequence DRB3-mRNA from Bos indicus (AccN X79346) and differs from it by 24 nucleotide substitutions which result in 16 amino acid substitutions. The peptide (on the basis of the reconstructed amino acid sequence) has 89% identity to the sequence encoded by the BIDRBF 188 locus (Bos indicus). The results obtained permit the sequence described by us to be considered as a new allele of the BoLA-DRB3 gene (DRB3.2 ^* X). The total frequency of the main six alleles (DRB3.2*8, *10, *11, *20, *34 and *X) occurring with a frequency of over 5% is about 60% in Iranian Sistani cattle. Fifteen alleles have <1% frequency. The highest frequency was observed for DRB3.2*8 allele (21.54%) like in other previously described breeds of Bos indicus (up to 23.07%). The Iranian breed Sistani has a high level of similarity by the spectrum of BoLA-DRB3 alleles and their frequencies to other Bos indicus breeds and significantly differs by these criteria from the Bos Taurus breeds. The Iranian Sistani herd under study includes alleles associated with to resistance to leukemia (DRB3.2*11 and *23) and to different forms of mastitis (DRB3.2* 2, *7, *11, *23 and *24) although their frequencies are low (from 0.77 to 5.37%). On the whole, a high level of diversity of BoLA-DRB3 gene alleles and the availability of alleles associated with resistance to different diseases makes this breed of interest for breeding practice. The article is published in the original.     

Genotyping the bovine kappa-casein locus using polymerase chain reaction]

Polymerase chain reaction (PCR) was used for detecting kappa-casein (kappa-casein) genotype in the synthetic breed (Jersey x Black and White x Holstein-Friesian) dairy cattle. The amplified 228 bp fragment includes a region, where relevant mutations lead to both the appearance of different kappa-casein alleles associated with amino acid substitutions and the appearance of new TaqI and HindIII restriction sites in ae-casein B gene. The specificity of the kappa-casein gene fragment amplification was supported by restriction analysis and Southern blot hybridization. Digestion of amplified fragment with endonucleases PstI, HindIII and/or TaqI allows detection of AA-, AB- and BB genotypes of kappa-casein. A total of 32 animals with known (18 samples) and unknown (14 samples) kappa-casein phenotypes were tested using PCR and blot hybridization. In all known cases the detected genotype confirmed the phenotype. Frequencies of the B allele and of the AB genotype in the breeding population are rather high (53.1 +/- 8.8 and 43.7%, respectively). The possibility of effective use of the PCR analysis for genotyping kappa-casein locus in bulls and their offspring has been shown. The advantages of the PCR method in large breeding programs and linkage analysis have been discussed.     

Analysis and Frequency of Bovine Lymphocyte Antigen (BoLA-DRB3) Alleles in Iranian Holstein Cattle

The bovine lymphocyte antigen (BoLA-DRB3) gene encodes cell surface glycoproteins that initiate immune response by presenting processed antigenic peptides to CD4 T helper cells. DRB3 is the most polymorphic bovine MHC class II gene which encodes the peptide-binding groove. DRB3 gene has been extensively evaluated as a candidate marker for association with various bovine diseases and immunological traits. This study describes genetic variability in the BoLA-DRB3 in Iranian Holstein cattle. This is the first study of the DNA polymorphism of the BoLA-DRB3 gene in Iranian Holstein cattle. Hemi-nested PCR-RFLP method is used for identification the frequency of BoLA-DRB3 alleles. The BoLA-DRB3 locus is highly polymorphic in the studied herd (26 alleles). Almost 67% of the alleles were accounted for four alleles (BoLA-DRB3.2*8, *24, *11, and *16) in Iranian Holstein cattle. The DRB3.2*8 allele frequency (26.6%) was higher than the others. The frequencies of the DRB3.2*54, *37, *36, *28, *25, *14, *13, *10, *1 alleles were lower than 1%. Significant distinctions have been found between Iranian Holstein cattle and other cattle breeds studied. In Iranian Holstein cattle the alleles (BoLA-DRB3.2*22, *2, and *16) associated with a lower risk of cystic ovarian disease in Holstein cattle are found. The alleles associated with the resistance to mastitis and to bovine leukemia virus infection BoLA-DRB3.2*11 and *23 are detected with the frequencies 10.4 and 4.4%, respectively. Thus, in the Iranian Holstein cows studied alleles associated with resistance to various diseases are found. The method of DNA-typing of animals can be used in agricultural practice for BoLA-DRB3 allele genotyping of cattle in order to reduce spreading of alleles providing susceptibility to mastitis or leukemia in cattle herds.__________From Genetika, Vol. 41, No. 6, 2005, pp. 817–822.Original English Text Copyright © 2005 by Nassiry, Eftekhar Shahroodi, Mosafer, Mohammadi, Manshad, Ghazanfari, Mohammad Abadi, Sulimova.The article was submitted by the authors in English.     

TGFα Reactivates Imprinted <Emphasis Type="Italic">Igf2</Emphasis> in the Parthenogenetic Mice Embryos and Placenta

Imprinted genes play important roles in the mammalian development. In the parthenogenetic embryos (PE), there is only expression of maternally expressed genes. Therefore, PEs are appropriate experimental models to study genomic imprinting controlling mechanisms. The maternally expressed H19 and paternally expressed Igf2 are reciprocally imprinted genes in normal embryos. Here, we studied effect of transforming growth factor alpha (TGFα) treatment in vitro (10 ng/ml at the morula stage) on the expression of Igf2/H19 locus in mice PE (9.5 days of gestation, 25 somites) and their placentas (PP). Using RT-PCR, we showed that TGFα reactivated maternally imprinted Igf2 gene in parthenogenetic embryos and placentas. In spite of similar Tgfα expression in the preimplantation stages, its expression in the 9.5-day parthenogenetic embryos is significantly less than in normal embryos (NE). In our experiments, it was shown that reactivation of Igf2 gene occurred independently of H19 gene. In vitro TGFα treatment of mouse PE reactivated paternally expressed Igf2 gene in the PE and PP. In the PE and PP, both Igf2 and H19 were expressed. It seems that TGFα can play an important role as modulator of the Igf2/H19 locus.