The bovine beta-lactoglobulin (LGB) gene is considered a potential quantitative trait locus in dairy cattle breeding. In Black-and-White dairy cattle the LGB gene has two predominant alleles A and B. This can result in three possible genotypes AA, AB and BB. Moreover, within the promoter of the gene several point mutations were found. A herd of one hundred and twenty-four Black-and-White cows were genotyped for two loci: locus LGB (exon IV, alleles A and B) and locus LGB-R (SSCP polymorphism within a fragment of LGB promoter: SSCP patterns R2, R3, R1, R9). In our sample 13 AA, 58 AB and 53 BB LGB cows and 66 R2, 16 R3, 40 R1 and 2 R9 LGB-R cows were identified. A statistical analysis revealed significant associations between LGB, LGB-R genotypes as well as intragenic haplotypes LGB/LGB-R and milk protein content during the first complete lactation. Cows with AA LGB genotype, R3 LGB-R SSCP pattern and AA/R3 haplotypes had the highest protein content. These results support the hypothesis that sequence variation within the promoter of the LGB gene is probably one of the factors responsible for differences in milk protein content.
Mitochondrial genetic system, comprising genome, transcription and translation processes play essential role in the function of mitochondria and thus for the survival of plants. The pathway from the genetic information encoded in the DNA to the functional protein leads through a very diverse RNA world. In this article, the current results obtained in the examination of plant mitochondrial transcription are described. Recent developments in the characterisation of promoter structure are presented.
The objective of this study was to search for polymorphisms and gene regulatory sequences in the 5' flanking region of the sheep insulin-like growth factor I (IGF-I) gene. PCR-SSCP analysis of the 5' flanking region revealed three banding patterns. Family study indicated that these patterns in mixed breed sheep corresponded with three genotypes (with their frequencies in parentheses) AA (0.70), AB (0.25), and BB (0.05), which arose from a one-locus, two allele (A, B) polymorphism. Genotypic frequencies in 22 purebred Polypay sheep were AA (0.77) and AB (0.23). Calculated frequency of the A allele in Polypays was 0.89. No deviation from Hardy-Weinberg equilibrium was detected in this study. Fragments amplified using DNA from homozygous individuals were sequenced and aligned next to each other. A T to C transition and a G to C transversion were found at positions 179 and 181, respectively, of the amplified PCR product, resulting in recognition sites for Bsp143II and HaeI. Analysis of a fragment of 2,162 base pairs upstream of Exon 1, assembled from sheep ESTs and sequence of our amplified PCR products, revealed a promoter sequence approximately 100 bp downstream of the polymorphic sites. The assembled DNA fragment shared 70% sequence homology between sheep and human. These results suggest that sequence of the 5' flanking region of IGF-I gene and location of the IGF-I promoters are similar in human and sheep.
In diverse eukaryotes, dsRNA triggers the destruction of mRNA sharing the same sequence as the dsRNA in the process called RNAi. The guides for sequence-specific degradation of mRNA are 21 nt short interfering RNAs (siRNAs). Synthetic siRNAs can efficiently mediate RNAi, but a drawback of RNAi is its transient nature as a result of the limited availability and stability of synthetic oligonucleotides. Recently, several groups reported the construction of expression plasmid vectors that mediate the production of siRNAs under control of Pol III promoters. These vectors allow the continued expression of siRNAs in the cells resulting in persistent and specific suppression of target genes. The retroviral siRNA expressing system allows for stable inactivation of the genes in primary cells or living organisms.
A biomarker, or molecular marker, or reporter gene is defined as a DNA sequence introduced into organisms. It confers a distinct genotype or phenotype to enable monitoring in a given environment. Molecular markers such as: LacZ (-galactosidase), xylE (catechol 2,3-dioxygenase), lux (bacterial luciferase), luc (insect luciferase), phoA (alkaline phosphatase), gusA and gurA (-glucuronidase), gfp (green fluorescent protein), bla (-lactamase) and antibiotic or heavy metals resistance genes are widely used in genetically engineered (GEMs) microorganisms research. These genes are involved in the detection and enumeration of GEMs after their introduction into the environment. Molecular markers, especially lux and gfp, are widely used in the creation of whole-cell based biosensors which are commonly used for the examination of toxicity of environmental pollutants.
During the past 15 years, the methylotrophic yeast Pichia pastoris has proven to be an excellent host for the production of both secreted and intracellular proteins. Numerous heterologous proteins have been produced at greater than gram per liter levels using alcohol oxidase promoter. The increasing popularity of this particular expression system can be attributed to several factors, most importantly: the simplicity of techniques for genetic manipulation, the ability to produce foreign proteins at high levels and the capability to perform many posttranslational modifications. The factors that drastically influence protein expression in this system include: copy number of the expression cassette, site and model of chromosomal integration of the heterologous gene, mRNA sequence and secondary structure, transcriptional and translational blocks, nature of secretion signal, endogenous proteases, host strain physiology, media and growth conditions. In this paper, I review how the system was developed, how it works and what can be done about it in the future.
Many laboratories worldwide are involved in the research on effective prevention and management of plant parasitic nematodes. Chemical control of these parasites is very costly and harmful to the environment, though the main strategy is to use resistance genes in breeding programs of crop plants. There is a limited number of naturally occurring resistance genes. Biotechnology can extend the usage of known resistance genes by transferring them to related and unrelated species via plant transformation. However, most promising is the development of new resistance strategies based on RNA interference or specific and inducible overexpression of nematocidal genes. Functional analysis of nematode and plant genes that are involved in induction and development of feeding structures can significantly help in engineering of new sources of resistance. Obviously, biotechnology is not the only prospective solution; however, it significantly enriches the breeders' toolbox. On the other hand, biotechnology-based pest management methods have been developed until recently, and often there are some shortcomings associated which require more research and optimization. Moreover, there is a permanent poor acceptance of genetically modified crops especially in Europe, which influences the decisions of policy makers. Nevertheless, recent genome scale experiments promise significant acceleration in the research and create a portfolio of numerous new possibilities.
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