A reliable method for sexing unincubated bird eggs for studying primary sex ratio

In birds, offspring sex ratio manipulation by mothers is now well established with potentially important consequences for evolution and animal breeding. In most studies on primary sex ratio of birds, eggs are sexed after incubation by the use of PCR methods targeted to the sex-linked CHD1 genes. Sexing of unincubated eggs would be preferred, but as fertile and infertile blastodiscs cannot be distinguished macroscopically, errors could arise from PCR amplifications of parental DNA associated with the vitelline membrane of infertile eggs. In this study, we stained blastodiscs without the vitelline membrane with Hoechst 33342. This allowed unequivocal distinction between fertile and infertile blastodiscs. Fertile blastodiscs contained thousands of fluorescent nuclei, whereas no nuclei were seen in infertile eggs. In addition, after nucleic acid analysis, fertile blastodiscs yielded much stronger chromosomal DNA and CHD1-targeted PCR bands on agarose gels compared with infertile blastodiscs. These findings indicate that fertile blastodiscs contain much more embryonic DNA than parental DNA, allowing reliable sexing of the fertile eggs. The differences between fertile and infertile blastodiscs in chromosomal DNA and CHD1 PCR banding intensities alone could also be used to distinguish fertile from infertile eggs without using Hoechst staining. We conclude that identifying fertile blastodiscs either by Hoechst staining or by analyzing the yield of chromosomal DNA and CHD1-PCR products, combined with CHD1-targeted PCR amplification, presents an easy and reliable method to sex unincubated eggs.     

Two gap junction channel (innexin) genes of the Bombyx mori and their expression

Gap junctions are clusters of intercellular channels that are associated with embryonic development and neural signaling. Innexins, invertebrate gap junction proteins, have been identified in Drosophila and Caenorhabditis. Here, we report the isolation and characterization of two novel members of the insect innexin family, Bm inx2 and Bm inx4, from embryos of the silkworm, Bombyx mori, during the germ-band formation stage. Bm inx2 is a single copy gene with one exon, while Bm inx4 is a single copy gene with four exons and three introns. The predicted proteins show structural similarities with other innexin family members, including four transmembrane (TM) domains, two extracellular loops (ELs), one cytoplasmic loop (CL), and typical conserved amino acids. Bm inx2 is phylogenetically orthologous to the other insect inx2 genes, but Bm inx4 is not orthologous to any known innexin including Dm inx4. Interestingly, Northern blotting and in situ hybridization showed that Bm inx2 was variously expressed across all developmental stages and in various tissues, with high expression seen in the nervous system at the time of embryogenesis. In contrast, Bm inx4 was transiently expressed at the germ-band formation stage of embryogenesis, and was specifically expressed in the ovary and testis during the larval and pupal stages. The isolation and characterization of these novel genes should form the basis for further study of the functional events that occur during development and neuronal communication in B. mori.     

Genetic Diversity of Pto-Like Serine/Threonine Kinase Disease Resistance Genes in Cultivated and Wild Strawberries

Degenerate oligonucleotide primers, designed based on conserved regions of several serine-threonine kinases (STK) previously cloned in tomato and Arabidopsis, were used to isolate STK candidates in wild and cultivated strawberries. Seven distinct classes of STKs were identified from three related wild species, i.e., Fragaria vesca, Fragaria chiloensis, and Potentilla tucumanensis, and seven different Fragaria x ananassa cultivars. Alignment of the deduced amino acid sequences and the Pto R protein from tomato revealed the presence of characteristic subdomains and conservation of the plant STK consensus and other residues that are crucial for Pto function. Based on identity scores and clustering in phylogenetic trees, five groups were recognized as Pto-like kinases. Strawberry Pto-like clones presented sequences that were clearly identified as the activation segments contained in the Pto, and some of them showed residues previously identified as being required for binding to AvrPto. Some of the non-Pto-like kinases presented a high degree of identity and grouped together with B-lectin receptor kinases that are also involved in disease resistance. Statistical studies carried out to evaluate departure from the neutral theory and nonsynonymous/synonymous substitutions suggest that the evolution of STK-encoding sequences in strawberries is subjected mainly to a purifying selection process. These results represent the first report of Pto-like STKs in strawberry.     

Problem of J-chain of immunoglobulins

Molecules of secretory immunoglobulins (Ig) of classes A and M (sIgA and sIgM) play the main role in protection of mucosae from pathogenic factors. The apparatus of synthesis of these molecules represent the most powerful part of the immune system. One of the key elements of the sIgA and sIgM is J-chain. It represents an acid polypeptide of molecular mass of about 15 kDa composed of 137 amino acid residues including 8 cysteine residues and one site of N-glycosylation. The primary structure of the J-chain is unique: attempts to ascribe it to any family of known proteins so far have failed. The J-chain is inserted into the sIgA and sIgM molecules by forming disulfide bonds with C-terminal sites of α-or μ-chains. It is necessary for formation of IgA dimers and IgM pentamers, for reception of these molecules by epithelial cells, binding of secretory component to them, and for transfer of sIgA and sIgM molecules onto mucosal surfaces and into secrets of exocrine glands. The J-chain has been revealed in the cytoplasm of the early T-and B-lymphocyte precursors not producing Ig. The J-chain is detected in the human embryonic liver cells earlier than the expression of the μ-chain gene begins. Study of mice with knockout of J-chain B-lymphocytes-producers has shown their block of functions of T-helpers providing formation of immunologic memory. Comparison of J-chain genes of mammals, amphibians, reptiles, and cartilaginous fishes has shown the degree of interspecies homology of these proteins to vary from 33% to 70%. The J-chain genes were revealed in representatives of all vertebrate classes except for cyclostomes and bony fishes. In 1996, data were published about the presence of the J-chain genes-homologs in invertebrates, tunicates, and cyclostomes. No papers reproducing or confirming these data have been published. On the contrary, in the literature an opinion appeared that indicate necessity to revise the notion about the presence of J-chain in invertebrates. The main unsolved issues on the J-chain involve the tertiary structure of this protein, its relation to some particular protein family, its functions in cells of the T-and B-lymphocytic differentiation lineages as well as its evolutionary age.     

Downregulation of RBSP3/CTDSPL, NPRL2/G21, RASSF1A, ITGA9, HYAL1, and HYAL2 in non-small cell lung cancer

Chromosomal and genome abnormalities of 3p are frequent in many epithelial tumors, including lung cancer. Several critical regions with a high frequency of hemi-and homozygous deletions in tumors are known for 3p, and more than 20 cancer-related genes occur in 3p21.3. Quantitative real-time PCR was used to measure the mRNA level for tumor-suppressor and candidate genes of 3p21.3 (RBSP3/CTDSPL, NPRL2/G21, RASSF1A, ITGA9, HYAL1, and HYAL2) in major types of non-small cell lung cancer (NSCLC): squamous cell lung cancer (SCC) and lung adenocarcinoma (AC). A significant (2-to 100-fold) and frequent (44–100%) decrease in mRNA levels was observed in NSCLC. The mRNA level decrease and its frequency depended on the histological type of NSCLC for all genes. The downregulation of RASSF1A and ITGA9 was significantly associated with AC progression; the same tendency was observed for RBSP3/CTDSPL, NPRL2/G21, HYAL1, and HYAL2. In SCC, the downregulation of all genes was not associated with the clinical stage, tumor cells differentiation, and metastasis in lymph nodes. The RBSP3/CTDSPL, NPRL2/G21, ITGA9, HYAL1, and HYAL2 mRNA levels significantly (5-to 13-fold on average) decreased at a high frequency (83–100%) as early as SCC stage I. Simultaneous downregulation of all six genes was observed in some tumor samples and was independent of the gene position in 3p21.3 and the functions of the protein products. The Spearman correlation coefficient r _s was 0.63–0.91, p < 0.001. The highest r _s values were obtained for gene pairs ITGA9-HYAL2 and HYAL1-HYAL2, whose products mediate cell-cell adhesion and cell-matrix interactions; coregulation of the genes was assumed on this basis. Both genetic and epigenetic mechanisms proved to be important for downregulation of RBSP3/CTDSPL and ITGA9. This finding supported the hypothesis that the cluster of cancerrelated genes in the extended 3p21.3 locus is simultaneously inactivated during the development and progression of lung cancer and other epithelial tumors. A significant and frequent decrease in the mRNA level of the six genes in SCC could be important for developing specific biomarker sets for early SCC diagnosis and new approaches to gene therapy of NSCLC.     

Genetic factors predisposing to a chronic course of virus hepatitis and liver fibrosis

The IL4 C(?590)T, IL4RA Ile50Val, and TNF G(?308)A polymorphisms were tested for association with the chronic development of virus hepatitis, the extent of which was inferred from the liver fibrosis stage. The frequency of allele A of the TNF G(?208)A polymorphism in patients with mild fibrosis was higher (24.5%) than in patients with moderate or severe fibrosis (13.4%) or cirrhosis (8.7%). The frequency of heterozygous genotype CT of the IL4 C(?590)T polymorphism significantly differed between cirrhosis (68.2%) and moderate or severe fibrosis (39.1%).