Fate of parental mitochondria in embryonic stem hybrid cells

In hybrid cells, not only are the nuclear genomes of parent cells fused, but their cytoplasm is as well. Mitochondrial DNA (mtDNA) is a convenient marker of cytoplasm that allows us to gain insight into the organization of hybrid-cell cytoplasm. We analyzed the parental mtDNA in hybrid cells resulting from the fusion of Mus musculus embryonic stem (ES) cells with splenocytes and fetal fibroblasts of DD/c mice or with splenocytes of M. caroli. Identification of parental mtDNA in hybrid cells was based on polymorphism among parental mtDNA for certain restriction endonucleases. We found that intra- and interspecific ES cell-splenocyte hybrid cells either entirely or partially lost mtDNA derived from a somatic partner, whereas ES cell-fibroblast hybrids retained mtDNA from both parents in similar ratios with a slight bias. The loss of somatic mitochondria by ES-splenocyte hybrids implies a nonrandom segregation of parental mitochondria, which was supported by a computer simulation of genetic drift. In contrast, ES cell-fibroblast hybrids show bilateral random segregation of the parental mitochondria judging from the analysis of mtDNA in single cells. Preferential segregation of somatic mitochondria does not depend on the differences in sequences of the parental mtDNA, but rather on the replicative state of parental cells.     

Evidence for tetrameric structure of mammalian hypoxanthine phosphoribosyltransferase

A fast electrophoretic variant of hypoxanthine phosphoribosyltransferase (HPRT) has been detected in Mus musculus bactrianus, a mouse subspecies from Middle Asia (USSR). The electrophoretic HPRT pattern yielded by hybrids between the somatic cell of LMTK^– (deficient in thymidine kinase) and the splenocytes of a male of M. m. bactrianus was five-banded. The pattern obtained from the germ cells of the ovaries from 14.5-day-old embryos from laboratory CBA mice × M. m. bactrianus crosses was also composed of five bands. The hybrids between the somatic cells of human fibroblasts × LMTK^– cells gave a three-banded electrophoretic HPRT pattern because the asymmetrical heteropolymeric isozymes are probably unstable. Taken together, all the evidence is in favor of a tetrameric structure of mammalian HPRT.     

Chromosome localization of the loci for PEPA,PEPB, PEPS, 1DH1, GSR,MPI, PGM1, NP,SOD1, and ME1 in the common shrew (Sorex araneus)

This report extends the genetic map of the common shrew (Sorex araneus) by adding chromosome assignments for ten genes to the seven already mapped (Pack et al. 1995). A somatic cell hybrid panel was used for the mapping. The genes for peptidase A (PEPA) and isocitrate dehydrogenase-1 (IDH1) map to chromosome de; the genes for phosphoglucomutase-1 (PGM1), superoxide dismutase-1 (SOD1), and mannosephosphate isomerase (MPI) are located on chromosome af; the genes for nucleoside phosphorylase (NP) and glutathione reductase (GSR) are on chromosome ik; and the genes for peptidase S (PEPS), malic enzyme-1 (ME1), peptidase B (PEPB) are found on chromosomes jl, go, and mp respectively. Received: 2 October 1995 / Accepted: 21 November 1995     

Methodological strategies for transgene copy number quantification in goats (Capra hircus) using real‐time PCR

Taking into account the importance of goats as transgenic models, as well as the rarity of copy number (CN) studies in farm animals, the present work aimed to evaluate methodological strategies for accurate and precise transgene CN quantification in goats using quantitative polymerase chain reaction (qPCR). Mouse and goat lines transgenic for human granulocyte‐colony stimulating factor were used. After selecting the best genomic DNA extraction method to be applied in mouse and goat samples, intra‐assay variations, accuracy and precision of CN quantifications were assessed. The optimized conditions were submitted to mathematical strategies and used to quantify CN in goat lines. The findings were as follows: validation of qPCR conditions is required, and amplification efficiency is the most important. Absolute and relative quantifications are able to produce similar results. For normalized absolute quantification, the same plasmid fragment used to generate goat lines must be mixed with wild‐type goat genomic DNA, allowing the choice of an endogenous reference gene for data normalization. For relative quantifications, a resin‐based genomic DNA extraction method is strongly recommended when using mouse tail tips as calibrators to avoid tissue‐specific inhibitors. Efficient qPCR amplifications (≥95%) allow reliable CN measurements with SYBR technology. TaqMan must be used with caution in goats if the nucleotide sequence of the endogenous reference gene is not yet well understood. Adhering to these general guidelines can result in more exact CN determination in goats. Even when working under nonoptimal circumstances, if assays are performed that respect the minimum qPCR requirements, good estimations of transgene CN can be achieved. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1390–1400, 2014     

Birth of normal kids after microinjection of pronuclear embryos in a transgenic goat (Capra hircus) production program in Brazil

This pilot project was designed to determine if normal kids could be produced after microinjection in pronuclear embryos and subsequent transfer to recipients in a transgenic goat program in Brazil. Twelve donors of the Saanen breed and 17 recipients of an undefined breed were used. The estrus of both donors and recipients was synchronized by a standard progestagen treatment and superovulation obtained by six pFSH injections. Donors in estrus were mated with fertile Saanen bucks. Zygotes were recovered surgically by flushing oviducts. The recovered zygotes with visible pronuclei were microinjected with 500 to 1000 copies of the human G-CSF gene. Two or four embryos were surgically transferred into the oviducts of recipients. One recipient became pregnant and two kids were born. No transgenic goat was identified after PCR analysis. Even though transgenic goats were not obtained, this experiment establishes the basis of a synchronization and superovulation regimen for use in goats raised in Brazil, for the purpose of collecting and manipulating the pronuclear embryos. This project also showed that microinjected one-cell goat embryos can survive to produce live young following surgical transfer.     

Bacterial formate dehydrogenase. Increasing the enzyme thermal stability by hydrophobization of alpha-helices

NAD+-dependent formate dehydrogenase (EC 1.2.1.2, FDH) from methylotrophic bacterium Pseudomonas sp.101 exhibits the highest stability among the similar type enzymes studied. To obtain further increase in the thermal stability of FDH we used one of general approaches based on hydrophobization of protein alpha-helices. Five serine residues in positions 131, 160, 168, 184 and 228 were selected for mutagenesis on the basis of (i) comparative studies of nine FDH amino acid sequences from different sources and (ii) with the analysis of the ternary structure of the enzyme from Pseudomonas sp.101. Residues Ser-131 and Ser-160 were replaced by Ala, Val and Leu. Residues Ser-168, Ser-184 and Ser-228 were changed into Ala. Only Ser/Ala mutations in positions 131, 160, 184 and 228 resulted in an increase of the FDH stability. Mutant S168A was 1.7 times less stable than the wild-type FDH. Double mutants S(131,160)A and S(184,228)A and the four-point mutant S(131,160,184,228)A were also prepared and studied. All FDH mutants with a positive stabilization effect had the same kinetic parameters as wild-type enzyme. Depending on the position of the replaced residue, the single point mutation Ser/Ala increased the FDH stability by 5-24%. Combination of mutations shows near additive effect of each mutation to the total FDH stabilization. Four-point mutant S(131,160,184,228)A FDH had 1.5 times higher thermal stability compared to the wild-type enzyme.     

Possible interaction mechanisms of allele and nonallele genes on the translation level]

Data on mechanisms of the formation of hybrid (heteropolymeric) isozymes are surveyed. Hybrid isozymes are considered to be the result of the interaction of allelic and some non-allelic genes at the translation level. Evidence is provided indicating that the formation of hybrid isozymes is closely related to the translation process of those mRNAs that synthesize allelic genes. It is suggested that the stage necessary for the formation of hybrid isozymes is compartmentalization (joint location) in the cytoplasm of the mRNAs that are synthesized by allelic genes. This suggestion helps to clarify many experimental data on the mechanisms of the formation of hybrid isozymes in various animal and plant species. One of the conclusions derived from this suggestion is that the compartmentalization of mRNAs of allelic genes creates conditions for an equal-probability quantitative manifestation of allelic genes in the phenotype prior to post-translation.     

The adrenal medulla and adrenergic innervation of the ventricles of the heart during the postnatal period of ontogenesis of the white rat (histofluorescent study)

The sympathetic innervation of the cardiac ventricles and dynamics of catecholamine contents in the adrenal medulla have been investigated in inbred white rats (1-3-week-old, immature, mature and old animals). During 1-3 weeks of age, development of adrenergic innervation of the heart is observed, in 1.5-month-old rats in approaches that in mature animals. In the old rats the arrangement density and fluorescent intensity of the adrenergic terminals of the cardiac ventricles in comparison to those in the mature animals decreases considerably. Catecholamine contents in the adrenal medulla in 3-week-old rats practically reaches those specific for mature animals. In the old animals the amount of catecholamines in the adrenal medulla decreases by 20%, comparing to those in the mature animals. Thus, maturation of the mediator link of the sympatho-adrenomedullary system is performed in it later than the hormonal one, while processes of old age involution are more intensive.