Of rabbit and man: Comparative gene mapping

Summary Nineteen cell hybrids were obtained by fusing rabbit (Oryctolagus cuniculus, OCU) fibroblasts and a Chinese hamster cell line HGPRT. Eleven enzymatic markers were investigated for cosegregation analysis. Seven could be assigned to OCU chromosomes: LDHA to OCU1; LDHB and TPI to OCU4; PEPB, NP, and ITP to OCU16; and G6PD to OCUX. Two assignments were considered possible: MDH2 to OCU15, and GUK to OCU3 or 15. Two could not be assigned: MDH1 and PGD. These results are consistent with the OCU-HSA chromosome homocologies previously reported, except for PEPB.     

Gene assignments and syntenic groups in the sacred baboon (Papio hamadryas)

Eighteen genes were assigned to chromosomes in the sacred baboon, Papio hamadryas, by their concordant segregation with the chromosomes in a set of baboon X Chinese hamster somatic cell hybrids. ACY1 was assigned to P. hamadryas chromosome 2 (PHA 2); SOD1 and MDH2 to PHA 3; ME1 and SOD2 to PHA 4; NP, MPI, PKM2, and HEXA to PHA 7; PP to PHA 9; ADA and ITPA to PHA 10; LDHB and TPI1 to PHA 11; MDH1 to PHA 13; ESD to PHA 17; and GPI and PEPD to PHA 20. Regional assignments were possible for ACY1 (PHA 2pter----q1) and MDH2 and SOD1 (PHA 3p). Five other independently segregating markers or syntenic groups (PGD, PGM1; and PEPC; PGM2 and PEPS; IDH1; LDHA and ACP2; and GSR) were also identified. Gene assignments and syntenic groups described in P. hamadryas are compared to those found in P. papio, the rhesus monkey, and man. A possible primate model for human lymphoid disease is discussed.     

An investigation of the homology of guanylate kinase isozymes in mammals and further evidence for multiple GUK gene loci

Guanylate kinase in the red cells of 63 different mammalian species was studied by electrophoresis and multiple molecular forms of the enzyme were found in all species. Two species were investigated in more detail. Using molecular weight estimates as a criterion of homology, the fallow deer and the Chinese hamster were found to have isozymes that corresponded to isozyme e, f, and g of man. Variation in the guanylate kinase isozymes was detected in a small population of orangutans. Results suggested that isozymes a and b were monomeric and that they were the products of a gene locus, GUK1, different from the locus GUK3 which coded for isozymes e, f, and g. Products c and d of the presumptive GUK2 locus were not found in the orangutan.     

Genomic regions influencing intramuscular fat in divergently selected rabbit lines

Intramuscular fat (IMF) is one of the main meat quality traits for breeding programmes in livestock species. The main objective of this study was to identify genomic regions associated with IMF content comparing two rabbit populations divergently selected for this trait, and to generate a list of putative candidate genes. Animals were genotyped using the Affymetrix Axiom OrcunSNP Array (200k). After quality control, the data involved 477 animals and 93 540 SNPs. Two methods were used in this research: single marker regressions with the data adjusted by genomic relatedness, and a Bayesian multiple marker regression. Associated genomic regions were located on the rabbit chromosomes (OCU) OCU1, OCU8 and OCU13. The highest value for the percentage of the genomic variance explained by a genomic region was found in two consecutive genomic windows on OCU8 (7.34%). Genes in the associated regions of OCU1 and OCU8 presented biological functions related to the control of adipose cell function, lipid binding, transportation and localisation (APOLD1, PLBD1, PDE6H, GPRC5D and GPRC5A) and lipid metabolic processes (MTMR2). The EWSR1 gene, underlying the OCU13 region, is linked to the development of brown adipocytes. The findings suggest that there is a large component of polygenic effect behind the differences in IMF content in these two lines, as the variance explained by most of the windows was low. The genomic regions of OCU1, OCU8 and OCU13 revealed novel candidate genes. Further studies would be needed to validate the associations and explore their possible application in selection programmes.     

Assignment of 6-phosphogluconate dehydrogenase and malate dehydrogenase to chromosome 3 ofAnopheles stephensi

Genetics and linkage analysis of 6-phosphogluconate dehydrogenase (6-PGD) and malate dehydrogenase (MDH) have been investigated in Anopheles stephensi. Both these markers were found to be autosomal and linked and have been assigned to linkage group III. Two mutant markers, Black larva (Bl) and golden-yellow larva (gy), were used to establish the map distances, and the current sequence of loci on chromosome 3 is as follows: Bl (3.75)-gy (14.53)-Mdh-2 (49.83)-6-pgd.     

Regulation of biosynthesis of secondary metabolites

Two partly purified malate dehydrogenase (EC 1.1.1.37) isoenzymes were isolated fromStreptomyces aureofaciens. This is the first example of a non-homogeneous enzyme in actinomycetes and one of the very few cases in bacteria in general. The characteristics of the enzymatic reaction were studied for each enzyme in relation to the concentration of both substrates and cofactors and the apparent Michaelis constant was calculated. It was found that the reaction was affected by Mg²⁺ ions and that SH-groups could be specifically inhibited. The optimal pH and the influence of temperature changes were also determined. In all the parameters, one of the isoenzymes resembled mitochondrial MDH, while the other resembel the supernatant MDH described in the literature in the tissues of higher organisms. The functional relationship of the two MDH isoenzymes inStreptomyces aureofaciens is discussed.     

Genome mapping of the silver fox. I. Determination of the chromosomal location of 8 fox genes and the search for homologous regions on fox and human chromosomes

Twenty-three silver fox-Chinese hamster somatic cell hybrids were analysed for the expression of fox enzyme loci and the segregation of fox chromosomes. This analysis made it possible to assign the gene PGD to chromosome 2, MDH2 to chromosome 3. NP to chromosome 10. APRT, ENO1, PGM1 to chromosome 12, MDH1 and IDH1 to chromosome 16. Possible use of the above-mentioned clone panel for fox gene mapping is analysed. An attempt to reveal homologous regions on fox and human chromosomes was made by comparative analysis of prometaphase fox and human chromosomes containing the homologous genes. The means and perspectives of verification of the hypothesis proposed are discussed.     

Partial 1H NMR assignments of the Escherichia coli dihydrofolate reductase complex with folate: evidence for a unique conformation of bound folate

Sequence-specific 1H assignments have been made for over 25% of the amino acid side chains of Escherichia coli dihydrofolate reductase complexed with folate by using a variety of two-dimensional techniques. Proton resonances were assigned by using a combination of site-directed mutagenesis and a knowledge of the X-ray crystal structure. Unique sets of NOE connectivities present in hydrophobic pockets were matched with the X-ray structure and used to assign many of the residues. Other residues, particularly those near or in the active site, were assigned by site-directed mutagenesis. The ability to assign unambiguously the proton resonances of these catalytically important residues allowed for extensive networks of NOE connectivities to follow from these assignments. As a consequence of these assignments, the orientation of the pterin ring of folate could be determined, and its conformation is similar to that of the productive dihydrofolate complex. Under these experimental conditions, only one bound form of the pterin ring could be detected.     

The effect of divergent selection for intramuscular fat on the domestic rabbit genome

An experiment of divergent selection for intramuscular fat was carried out at Universitat Politècnica de València. The high response of selection in intramuscular fat content, after nine generations of selection, and a multidimensional scaling analysis showed a high degree of genomic differentiation between the two divergent populations. Therefore, local genomic differences could link genomic regions, encompassing selective sweeps, to the trait used as selection criterion. In this sense, the aim of this study was to identify genomic regions related to intramuscular fat through three methods for detection of selection signatures and to generate a list of candidate genes. The methods implemented in this study were Wright’s fixation index, cross population composite likelihood ratio and cross population – extended haplotype homozygosity. Genomic data came from the 9th generation of the two populations divergently selected, 237 from Low line and 240 from High line. A high single nucleotide polymorphism (SNP) density array, Affymetrix Axiom OrcunSNP Array (around 200k SNPs), was used for genotyping samples. Several genomic regions distributed along rabbit chromosomes (OCU) were identified as signatures of selection (SNPs having a value above cut-off of 1%) within each method. In contrast, 8 genomic regions, harbouring 80 SNPs (OCU1, OCU3, OCU6, OCU7, OCU16 and OCU17), were identified by at least 2 methods and none by the 3 methods. In general, our results suggest that intramuscular fat selection influenced multiple genomic regions which can be a consequence of either only selection effect or the combined effect of selection and genetic drift. In addition, 73 genes were retrieved from the 8 selection signatures. After functional and enrichment analyses, the main genes into the selection signatures linked to energy, fatty acids, carbohydrates and lipid metabolic processes were ACER2, PLIN2, DENND4C, RPS6, RRAGA (OCU1), ST8SIA6, VIM (OCU16), RORA, GANC and PLA2G4B (OCU17). This genomic scan is the first study using rabbits from a divergent selection experiment. Our results pointed out a large polygenic component of the intramuscular fat content. Besides, promising positional candidate genes would be analysed in further studies in order to bear out their contributions to this trait and their feasible implications for rabbit breeding programmes.     

Physical assignments of 68 porcine cosmid and lambda clones containing polymorphic microsatellites

Two lambda phage and 66 cosmids containing informative porcine microsatellites were assigned to 17 of 18 porcine autosomes and the X Chromosome (Chr) by fluorescence in situ hybridization (FISH). These assignments provide additional physically anchored markers to integrate the porcine physical and genetic maps. Received: 2 October 1995 / Accepted: 12 December 1995     

Gene mapping in the Chinese hamster and conservation of syntenic groups and Q-band homologies between Chinese hamster and mouse chromosomes

Using Chinese hamster/mouse somatic cell hybrids segregating hamster chromosomes, we assigned 15 enzyme genes to six different Chinese hamster autosomes. Of the 15 loci, three genes, HK1, PEPC, and SORD, were newly assigned to chromosomes 1, 5, and 6, respectively, while ENO1, PGD, and PGM1 were assigned to the long arm of chromosome 2, in the segment 2q113----qter. The locations of the following loci were confirmed: ESD, NP, and PEPB on chromosome 1, ME1 and MPI on chromosome 4, AK1 on chromosome 6, and GPI and PEPD on chromosome 9. Comparative mapping of Chinese hamster and laboratory mouse chromosomes revealed conservation of syntenic groups and extensive banding homology between the Chinese hamster and mouse chromosomes on which homologous enzyme markers have been mapped.     

Malate dehydrogenase (MDH; EC 1.1.1.37) isozymes in tissues and callus cultures ofCereus peruvianus (cactaceae)

Malate dehydrogenase (MDH; EC 1.1.1.37) isozymes were investigated in seeds and in seedlings and calli cultures ofC. peruvianus to determine if the changes in MDH isozyme banding patterns could be used as biochemical markers to identify the origin of regenerated plants from callus tissues. Four cytoplasmic MDH isozymes (sMDH), five mitochondrial MDH isozymes (mMDH), and one glyoxysomal MDH isozyme (gMDH) were detected and showed tissue- and stage-specific expression. A relationship of mMDH and gMDH isozyme patterns with callus tissues subcultured in three hormonal combinations and with the plants regenerated from these callus tissues was demonstrated. Furthermore, temperature and mechanical stress were found to be closely related to mMDH-1 activity in callus culture. Therefore, the different patterns of MDH isozymes in the various tissues ofC. peruvianus can be used as biochemical markers for the study of gene expression during development and as powerful tools in monitoring studies on callus cultures. This research was supported by the CNPq.     

Characteristics of somatic cell hybrids (mouse X Chinese hamster) with different ratios of parental species chromosome sets. IV. Electrophoretic analysis of several enzymes of the dehydrogenase class]

Electrophoretic mobilities in polyacrylamide gel of five dehydrogenases: NADP-dependent malate dehydrogenase (NADP-MDH), 6-phosphogluconate dehydrogenase (6PGD), alcohol dehydrogenase (ADH), glucose-6-phosphate dehydrogenase (G6PD) and glutamate dehydrogenase (GDH) were investigated in a series of mouse X Chinese hamster somatic cell hybrids. Seven hybrid lines with different ratio of chromosome sets of hamster and mouse: 1:1, 2:1, 3:1 and 1:2 respectively were studied. NADP-MDH and 6PGD of both parental species and intermediate hybrid bands were present in all hybrids except two lines. These lines had only hamster MDH due to the elimination of mouse chromosomes. A correlation was found between the gene dose and the intensity of the expression of the MDH bands. The mouse type ADH was detected in all hybrids. The hamster ADH was found in one of the hybrid lines that lost all mouse chromosomes during cultivation. It is suggested that hamster ADH activity was suppressed in hybrids by the mouse genome. The species origin of GDH and G6PD could not be established due to similarity of electrophoretic mobilities of respective enzymes in parental cells.     

Malate Dehydrogenase from the Thermophilic Bacterium <Emphasis Type="Italic">Vulcanithermus medioatlanticus</Emphasis>

Thermostable dimeric malate dehydrogenase (MDH) was isolated from the microorganism of hydrothermal vents Vulcanithermus medioatlanticus. The enzyme was electrophoretically homogeneous and possessed the specific activity of 6.9 U/mg. The large molecular weight of the subunits (55 kD) is likely to provide the rigidity of the enzyme structure (the activation energy of the enzymatic reaction is 32.6 kJ/mol). The thermophilic MDH differs little from the mesophilic enzyme in terms of kinetic and regulatory characteristics.     

New gene assignments and syntenic groups in the baboon (Papio papio)

MDH2, SOD2, PEPS, and ITPA were assigned to Papio papio chromosomes 3, 4, 5, and 10, respectively, by their concordant segregation with previously assigned gene markers in a set of baboon X mouse somatic cell hybrids. The linkage of NP, IDH2, SORD, MPI, and PKM2 was confirmed, and three other independently segregating markers (MDH1, ACY1, and PEPB) were identified. Syntenic groups described in the baboon are compared to those found in man and in the rhesus monkey.     

Aanlysis of dCMP deaminase and CDP reductase levels in hamster cells infected by herpes simplex virus.

Several enzymatic activities involved in the biosynthetic pathways of nucleotides, including thymidine kinase, which has been used as a biochemical marker in studies of gene transfer, are induced by herpes simplex virus (HSV). The utility of additional markers prompted us to reanalyze the effects of HSV infection on the activities of two other enzymes for which direct selective methods can be devised: dCMP deaminase and CDP reductase. For this purpose, mutant Chinese hamster (lA1) cells devoid of dCMP deaminase activity or Syrian hamster (BHK-21/C13) cells were infected by HSV type 1 or 2, and the activities of thymidine kinase, dCMP deaminase, and CDP reductase were measured in the cell extracts. The reported induction of thymidine kinase and CDP reductase by HSV was confirmed, whereas the stimulation of dCMP deaminase activity could not be observed. For both cell lines, the HSV-induced CDP reductase differed from the host enzyme by sensitivity to inhibition by both dTTP and dATP. This property should be helpful in developing a selection system for this activity.     

Sequence studies of rabbit serum transferrin

Large fragments of rabbit serum transferrin have been prepared by enzymatic digestion with subtilisin and by chemical cleavage with BNPS-skatole. Sequence determinations on fragments from the N-terminal lobe lead to the assignment of 273 residues and those from the C-terminal lobe 267 residues. Together with previous determinations, a total of 614 of the ca 679 residues in rabbit transferrin have been assigned. A number of corrections are made to the preliminary sequence assignments of O.U. Beg, H.A. McKenzie and D.C. Shaw (1988) Biochemistry International 17, 1135-1142.     

Interstitial and parenchymal cells in the pineal gland of the golden hamster

Summary A combined thin-section/freeze-fracture study was performed on the superficial pineal gland of the golden hamster, comparing the parenchymal and interstitial cells of this animal with those previously investigated in rats. In contrast to rats, no gap junctions and gap/tight junction combinations could be found between pineal parenchymal cells of the hamster. Furthermore, the interstitial cells of the hamster pineal gland were found to have large flat cytoplasmic processes, which abut over large areas equipped with tight junctions. In thin sections, profiles of interstitial cell processes were seen to surround groups of pinealocytes. Interstitial cells and their sheet-like, tight junction-sealed processes thus appear to delimit lobule-like compartments of the hamster pineal gland. Because the classification of the interstitial cells is uncertain, the expression of several markers characteristic of mature and immature astrocytes and astrocyte subpopulations has been investigated by indirect immunohistology. Many of the non-neuronal elements in the pineal gland are vimentin-positive glial cells, subpopulations of which express glial fibrillary acidic protein (GFA) and C1 antigen. The astroglial character of these cells is supported by the lack of expression of markers for neuronal, meningeal and endothelial cells. M1 antigen-positive cells have not been detected.Supported by a grant from Deutsche Forschungsgemeinschaft (Scha 185/9-2)     

Assignment of 204 genes localized on HSA17 to a porcine RH (IMpRH) map to generate a dense comparative map between pig and human/mouse

Bi- and uni-directional chromosome painting (ZOO-FISH) and gene mapping have revealed correspondences between human chromosome (HSA) 17 and porcine chromosome (SSC) 12 harboring economically important quantitative trait loci. In the present study, we have assigned 204 genes localized on HSA17 to SSC12 to generate a comprehensive comparative map between HSA17 and SSC12. Two hundred fifty-five primer pairs were designed using porcine sequences orthologous with human genes. Of the 255 primer pairs, 208 (81.6%) were used to assign the corresponding genes to porcine chromosomes using the INRA-Minnesota 7000-rad porcine x Chinese hamster whole genome radiation hybrid (IMpRH) panel. Two hundred three genes were integrated into the SSC12 IMpRH linkage maps; and one gene, PPARBP, was found to link to THRA1 located in SSC12 but not incorporated into the linkage maps. Three genes (GIT1, SLC25A11, and HT008) were suggested to link to SSC12 markers, and the remaining gene (RPL26) did not link to any genes/expressed sequence tags/markers registered, including those in the present study. A comparison of the gene orders among SSC12, HSA17, and mouse chromosome 11 indicates that intra-chromosomal rearrangements occurred frequently in this ancestral mammalian chromosome during speciation.