Observations on bacteria associated with pigeon crop

Lactobacilli, streptococci and coliforms of pigeon crop contents, 5th wash, macerate and those of first-day pigeon milk have been studied. Streptococci predominated in all the samples tested. Relatively higher counts of lactobacilli and streptococci in crop macerate than in the 5th wash suggested the adhesion of these bacteria to crop wall. Because of frequent occurrence in crop of partially digested fibrous food, its contents were tested for the presence of cellulolytic bacteria. The results indicated that four isolates were capable of utilizing cellulose with the resultant production of reducing sugars. It is inferred that the crop microflora is involved in the degradation of dietary fibre in the pigeon.     

Expression of cloned genes in pro- and eukaryotic cells

Expression of cloned genes in new environment is reviewed. Gene expression is possible under control of their own regulatory elements in the cells of related organisms. Genes may also function in cells of taxonomically remote organisms; for example, the genes of lower eukaryotes are active in bacterial cells, the Drosophila gene -- in yeast cells. The main principles of construction of pro- and eukaryotic vectore capable to provide the expression of DNA sequences in corresponding recipient cells are discussed.     

Structure and expression of cloned murine IFN-alpha genes

The mouse has an interferon-alpha (MuIFN-alpha) gene family containing at least four, and likely more than ten members. A segment of mouse chromosomal DNA and cDNAs encoding murine alpha IFNs have been cloned, and the sequence of two MuIFN-alpha DNAs determined. No intron was found in the chromosomal gene. The two coding sequences produced biologically active IFN when expressed in monkey cells under the control of an SV40 promoter, and in E.coli under the control of the ampicillinase promoter. MuIFN-alpha 1 had no detectable activity on human cells, while MuIFN-alpha 2 was 20% as active on human as on mouse cells.     

Sequence organization of cloned intracisternal A particle genes

Seven recombinant DNA clones containing mouse intracisternal A particle genes were isolated and analyzed by restriction enzyme digestion, Southern blot analysis and heteroduplex mapping. The sequence organization of the individual genes was found to differ, with one end of the gene region being most variable, while a central segment of 1.8 kb was missing from two of the clones. A third region, common to all the clones and containing the 3' end of the gene, is present in about 1800 copies per haploid genome, but the central portion is found in only 650 copies. The same reiteration frequency is found in both myeloma tumor and mouse liver DNA. The most abundant intracisternal A particle RNA in two different myeloma lines was found to be 3.5 kb, and RNA/DNA hybrids show that the RNA is homologous to all but a small internal segment of one of the clones.     

Cloning and sequencing of quail and pigeon prion genes

Quail and pigeon PrP genes were cloned and sequenced. Like mammalian PrP genes, quail and pigeon genes are encoded by a single exon of a single copy gene in the genome. All of the structural features of mammalian PrP genes were found in the quail and pigeon PrP gene. Compared with the nucleotide sequences of mammalian PrP, they display generally 30% similarity. When compared with chicken PrP's DNA sequence, they show a higher homology (90%), and an even higher homology (99%) when compared to each other. A phylogenetic tree was constructed to trace the evolution of the prion gene in animals.     

Molecular mapping and tagging of genes in crop plants

In India, molecular mapping and tagging of agronomically important genes using RFLP and RAPD markers have been carried out in three different crops: rice, mustard and chickpea. In rice, tagging of genes for resistance to gall midge and blast has been accomplished. Molecular mapping of cooking quality traits in rice is in progress. For fingerpringting rice cultivars, suitable probe enzyme combinations have been identified. In mustard, a partial RFLP linkage map has been constructed and one of the yellow seed-coat colour loci has been mapped. Significant associations of RFLP markers with quantitative traits have also been established. Potential use of RAPD markers to identify heterotic groups among mustard accessions has been demonstrated. In chickpea, the occurrence of considerable interspecific DNA polymorphism as revealed by RAPD analysis has facilitated construction of a partial linkage map.     

Regulation of alpha-ketoglutarate dehydrogenase complex from pigeon breast muscle]

The activity of alpha-ketoglutarate dehydrogenase complex from pigeon breast muscle is controlled by ADP and the reaction products, i. e. succinyl-CoA and NADH. ADP activates the alpha-ketoglutarate dehydrogenase component of the complex, whereas NADH inhibits alpha-ketoglutarate dehydrogenase and lipoyl dehydrogenase. In the presence of NADH the kinetic curve of the complex with respect to alpha-ketoglutarate and NAD and the dependence of upsilon versus [NAD] and upsilon versus [Lip (SH)2] in the lipoyl dehydrogenase reaction are S-shaped. In the absence of inhibitor ADP had no activating effect on lipoyl dehydrogenase; however, in the presence of NADH ADP decreases the cooperativity for NAD. The cooperative kinetics of the constituent enzymes of the complex are indicative of its allosteric properties. Isolation of the alpha-ketoglutarate dehydrogenase complex and its lipoyl dehydrogenase and alpha-ketoglutarate dehydrogenase components in a desensitized state confirms their allosteric nature. It is assumed that NADH effects of isolated alpha-ketoglutarate dehydrogenase is due to a shift in the equilibrium between different oligomeric forms of the enzyme.     

Genetic screen for cloned release factor genes.

Nonsense suppression reflects competition between a nonsense suppressor tRNA and a translational release factor. This provides a simple way to screen for release factor genes cloned into a multicopy plasmid. We have confirmed that the expected competition occurs with the gene for release factor 2, cloned by C.T. Caskey et al. (C.T. Caskey, W.C. Forrester, W. Tate, and C.D. Ward, J. Bacteriol. 158:365-368, 1984), and used it to clone the gene for release factor 1.     

Development of broad-host-range vectors for expression of cloned genes in Pseudomonas

The cloning and expression of genes in Pseudomonas have been difficult, until now, due to the absence of vector systems that contain multiple restriction sites downstream from promoter sequences that are functional in Pseudomonas. We report here the construction of several broad-host-range vectors that can be utilized in either Pseudomonas or Escherichia coli and that rely on easily selectable antibiotic resistance markers with multiple cloning sites. These vectors were constructed by inserting the entire pUC13 sequence into derivatives of the RSF1010 wide-host-range plasmid. From this construction, other derivatives were obtained, specifically a lacZ::KmR fusion gene which provides an easily selectable marker in both E. coli and Pseudomonas. These vectors have been used to express the Pseudomonas putida cytochrome P450 monoxygenase gene in a P450-deficient P. putida strain. Thus, these vectors allow for the cloning, expression and selection of Pseudomonas genes in Pseudomonas by complementation.     

The role of cloned genes in the prevention of genetic disease

The application of recombinant DNA technology to the study of human genetic disease promises to increase the scope for carrier detection and prenatal diagnosis. Here we summarize current experience with prenatal diagnosis of single-gene disorders by DNA analysis and highlight some of the technical and organizational problems that remain to be solved.