Development of Protocol for Production of Primary Antibody against Ovalbumin Protein in Chicken for Detection of the Protein through Western Blotting

Ovalbumin, a major protein of egg white plays many roles including providing nutrition to the developing embryo, acting as coagulating agent, folliculogenesis and angiogenesis in chicken and other animals. This protein is expressed mainly in magnum and then deposited over the yolk of the oocyte/zygote. Hence, it is important in formation of egg and is an essential target to measure. We cloned chicken ovalbumin CDS in pAcGFP-C1 vector and has been initially expressed in chicken primary magnum cell culture. The ovalbumin protein tagged with 6x Histidine was purified from cell culture and used for production of primary antibody in rat. The ovalbumin protein along with freund’s adjuvant was injected to the rat, booster was given, and finally, hyper-immune sera was collected from rat. The antisera was purified for isolation of IgG. The IgG was used as primary antibody for Western blotting. Through Western blotting, ovalbumin protein isolated from chicken magnum was detected and the protocol was established to detect chicken ovalbumin protein.

Drosophila simulans Lethal hybrid rescue mutation (Lhr) rescues inviable hybrids by restoring X chromosomal dosage compensation and causes fluctuating asymmetry of development.

The Drosophila simulans Lhr rescues lethal hybrids from the cross of D. melanogaster and D. simulans. We describe here, the phenotypes of Lhr dependent rescue hybrids and demonstrate the effects of Lhr on functional morphology of the salivary chromosomes in the hybrids. Our results reveal that the phenotypes of the 'Lhr dependent rescued' hybrids were largely dependent on the genetic background and the dominance in species and hybrids, and not on Lhr. Cytological examination reveal that while the salivary chromosome of 'larval lethal' male carrying melanogaster X chromosome was unusually thin and contracted, in 'rescued' hybrid males (C(mel)X(mel)Y(sim); A(mel)A(sim)) the X chromosome showed typical pale staining, enlarged diameter and incorporated higher rate of (3)H-uridine in presence of one dose Lhr in the genome. In hybrid males carrying simulans X chromosome (C(mel)X(sim)Y(mel); A(mel)A(sim)), enlarged width of the polytene X chromosome was noted in most of the nuclei, in Lhr background, and transcribed at higher rate than that of the single X chromosome of male. In hybrid females (both viable, e.g., C(mel)X(mel)X(sim); A(mel)A(sim) and rescued, e.g., C(mel)X(mel)X(mel); A(mel)A(sim)), the functional morphology of the X chromosomes were comparable to that of diploid autosomes in presence of one dose of Lhr. In hybrid metafemales (C(mel)X(mel)X(mel)X(sim); A(mel)A(sim)), two dose of melanogaster X chromosomes and one dose of simulans X chromosome were transcribed almost at 'female' rate in hybrid genetic background in presence of one dose of Lhr. In rescued hybrid males, the melanogaster-derived X chromosome appeared to complete its replication faster than autosomes. These results together have been interpreted to have suggested that Lhr suppresses the lethality of hybrids by regulating functional activities of the X chromosome(s) for dosage compensation.

Binding affinity of leucine-containing chromatin proteins to the polytene X chromosome of Drosophila hydei and its significance.

Association of newly synthesised non-histone chromosomal protein to the polytene X chromosome of larval salivary glands of D. hydei has been examined by autoradiographic procedure using 3H-leucine. It has been observed that 3H-leucine labelling pattern is inhibited in presence of puromycin. Results further reveal that there is a reasonable concordance between the binding affinity of newly synthesized protein on the single X chromosome of the male and paired X's of the female. A sitewise analysis of 3H-leucine labelling reveals that although 3H-leucine incorporation pattern are not strictly comparable with 3H-RNA synthesis pattern observed under in vivo transcription condition, the labelling pattern with 3H-leucine are not merely the reflection of mass distribution of protein in the polytene chromosomes. Certain aspects of regulation in the organisation of male and female X chromosome in Drosophila by de novo synthesis of protein are discussed.

Mosaic pattern of X-chromosomal transcription in a strain of Drosophila melanogaster with aneuploid X chromosome.

Organisation and template activity pattern of salivary gland chromosomes of a segmental male aneuploid of D. melanogaster, carrying duplication for the segment 8C-20F of X chromosome, have been examined by in situ transcription. In an earlier study [Chatterjee, Chromosoma 91 (1985) 259], it was suggested that in male aneuploids, up to an additional length of 8C-20F, the template activity of X chromosome tends to remain at a male level and beyond that level shifts towards female level. A large scale search of the template activity pattern of the aneuploid carrying dp.(8C-20F) clearly indicates that presence of the duplication fragment to X in the normal karyotype (1X2A) lead to a varying degree of condensation of euchromatic regions of entire X chromosome (X + X fragment 8C-20F) starting from 'male' level, over a wide range of 'intermediate' level to a normal 'female' level. In this study, the individual cells of the aneuploid appeared to display their own state of X condensation and transcription. Although in the aneuploid, X chromosomal activity is not determined by a purely quantitative effect of X vs. autosomal material (X:A ratio = 0.81), the 8C-20F segment of X chromosome must contain some major elements concerned with the signal given by X:A ratio for X chromosome differentiation.