Further investigation on nuclear transplantation in different orders of teleost: the combination of the nucleus of Tilapia (Oreochromis nilotica) and the cytoplasm of Loach (Paramisgurnus dabryanus).

The nucleus of a blastula cell from Tilapia (Oreochromis nilotica, family Cichlidae, order Perciformes) was transplanted into an enucleated egg of Loach (Paramisgurnus dabryanus, family Cobitidae, order Cypriniformes). From among 3747 nucleo-cytoplasmic hybrid (NCH) eggs two NCH larval fish (0.05%) were obtained; one died on the 6th day and the other died on the 12th day after the operation. Morphological examinations showed that both NCH larval fish had developed normally with an opened mouth except they could not take food after complete utilization of their egg yolk on the 5th day of development. The possible mechanisms for obtaining such inter-order NCH larval fish are discussed. This is the first report indicating that inter-order NCH larval fish can be obtained in spite of their evolutionary divergence.     

Genetic and molecular analysis of spontaneous respiratory deficient (res-) mutants of Escherichia coli K-12

Respiratory deficient (res-) mutants of E. coli are slow growing microcolonial, anaerobic, catalase and benzidine negative strains whose broad phenotypic alteration may result from pleiotropic mutations in genes of the hemin biosynthetic pathway. They are easily recovered from platings of sensitive cells on concentrations of gentamicin higher than the minimal inhibitory concentration. These mutants show a dramatic change in their biochemical diagnostic profile resulting primarily from deficiencies in the active transport mechanisms of the cell. Using well-marked F- and Hfr strains, 157 mutants were analyzed from 3 different parent strains; all but 2 resulted from mutations in 3 loci of the hemin biosynthetic pathway. Of these a marked skew to hemB- mutations was seen, with more than 80% mapping there. The possibility that this hot spot resulted from transpositional activity was tested by Southern hybridization of EcoRI digests of the chromosomal DNA, using as a probe, a 2.8-kb fragment containing the hemB gene. The WT and other hemB+ control strains contained a 14.6-kb fragment. Of 18 hemB strains tested, 14 showed deletion and insertion mutations which fell into four classes based on the variation in the size of the fragment or on the absence of hybridization. The latter resulted from complete deletion of the hemB gene. An increase in fragment size from 1.5-kb to 3.4-kb was observed in some of the strains.     

Multiple uses of liquepipets in Southern, northern, and western blots

Individually wrapped, sterile disposable transfer pipets can be used in the isolation of ds-DNA and ds-RNA fragments from gels as well as in the screening of multiple samples in Southern, Northern, and Western blots without potential contamination by exogenous nucleases and proteases. The sensitivity and results obtained by this method are comparable to those obtained by conventional methods. All the prehybridization, blocking, hybridization, and detection processes can be performed within the transfer pipet. The isotopically labeled probes used in hybridization can easily be recovered, stored for reuse, or disposed of as waste with no potential contamination of personnel or laboratory equipment. Strip blots are stable in appropriate buffers within the liquepipets which can be shipped easily worldwide for comparative analyses by collaborative investigators. This method is simple, time saving, and inexpensive and is particularly suitable for multiple sample screening. Other potential applications of this procedure are discussed.     

Site-specific integration and expression of a developmental promoter in Myxococcus xanthus.

A series of intercellular signals are involved in the regulation of gene expression during fruiting body formation of Myxococcus xanthus. Mutations which block cell interactions, such as csgA (formerly known as spoC), also prevent expression of certain developmentally regulated promoters. csgA+ cells containing Tn5 lac omega DK4435, a developmentally regulated promoter fused to lacZ, began synthesizing lacZ mRNA 12 to 18 h into the developmental cycle. beta-Galactosidase specific activity increased about 12 h later. Neither lacZ mRNA nor beta-galactosidase activity was detected in a developing csgA mutant containing omega DK4435. The developmental promoter and its fused lacZ reporter gene were cloned into a pBR322-derived plasmid vector containing a portion of bacteriophage Mx8. These plasmids preferentially integrated into the M. xanthus chromosome by site-specific recombination at the bacteriophage Mx8 attachment site and maintained a copy number of 1 per chromosome. The integrated plasmids were relatively stable, segregating at a frequency of 0.0007% per generation in the absence of selection. The cloned and integrated promoter behaved like the native promoter, expressing beta-galactosidase at the proper time during wild-type development and failing to express the enzyme during development of a csgA mutant. The overall level of beta-galactosidase expression in merodiploid cells containing one native promoter and one promoter fused to lacZ was about half that of cells containing a single promoter fused to lacZ. These results suggest that the timing of developmentally regulated gene expression is largely independent of the location of this gene within the chromosome. Furthermore, they show that site-specific recombination can be a useful tool for establishing assays for promoter or gene function in M. xanthus.     

2- and 8-azido photoaffinity probes. 1. Enzymatic synthesis, characterization, and biological properties of 2- and 8-azido photoprobes of 2-5A and photolabeling of 2-5A binding proteins

The 2- and 8-azido trimer 5'-triphosphate photoprobes of 2-5A have been enzymatically synthesized from [gamma-32P]2-azidoATP and [alpha-32P]8-azidoATP by 2-5A synthetase from rabbit reticulocyte lysates. Identification and structural determination of the 2- and 8-azido adenylate trimer 5'-triphosphates were accomplished by enzymatic hydrolyses with T2 RNase, snake venom phosphodiesterase, and bacterial alkaline phosphatase. Hydrolysis products were identified by HPLC and PEI-cellulose TLC analyses. The 8-azido photoprobe of 2-5A displaces p3A4[32P]pCp from RNase L with affinity equivalent to p3A3 (IC50 = 2 X 10(-9) M in radiobinding assays). The 8-azido photoprobe also activates RNase L to hydrolyze poly(U) [32P]pCp 50% at 7 X 10(-9) M in core-cellulose assays. The 2- and 8-azido photoprobes and authentic p3A3 activate RNase L to cleave 28S and 18S rRNA to specific cleavage products at 10(-9) M in rRNA cleavage assays. The nucleotide binding site(s) of RNase L and/or other 2-5A binding proteins in extracts of interferon-treated L929 cells were investigated by photoaffinity labeling. Dramatically different photolabeling patterns were observed with the 2- and 8-azido photoprobes. The [gamma-32P]2-azido adenylate trimer 5'-triphosphate photolabels only one polypeptide with a molecular weight of 185,000 as determined by SDS gel electrophoresis, whereas the [alpha-32P]8-azido adenylate trimer 5'-triphosphate covalently photolabels six polypeptides with molecular weights of 46,000, 63,000, 80,000, 89,000, 109,000, and 158,000. Evidence that the photolabeling by 2- and 8-azido 2-5A photoprobes was highly specific for the p3A3 allosteric binding site was obtained as follows.(ABSTRACT TRUNCATED AT 250 WORDS)     

Computer aided analysis for biosensing and screening

Biosensors with animal and microbial cells immobilized close to the tip of a membrane electrode have been developed for chemical and drug testing. Our experimental results show that biosensors can be used for drug screening and to provide useful information about various cell-chemical interactions. A computer aided analysis (CAA) software package is being developed here using the biosensor for various screening purposes. This software package enables us to use a computer to analyze the biosensor dynamic responses. Computer simulation and parameter estimation techniques are used to select the best model and to describe the biochemical and pharmacologic effects of various chemicals and drugs on different cell lines.