Restriction fragment length polymorphism of ovine casein genes: close linkage between the αs1-,αs2-,ß- and k-casein loci

Restriction fragment length polymorphism (RFLP) of ovine casein genes was investigated. Genomic DNA from 56 rams was digested with 10 restriction endonucleases and Southern blots probed with the four ovine casein cDNAs (alpha s1-, beta-, alpha s2- and kappa-Cn). Five enzymes, namely, BglI, PvuII, RsaI, TaqI and HindIII revealed nine different RFLPs. The inheritance of six of these polymorphisms was studied by segregation analysis of gametes in nine rams' families, and each of them could be related to the existence of alleles at the relevant casein locus. A close linkage between the four ovine casein genes was demonstrated since no recombination within the four pairs of loci examined, alpha s1-beta-Cn, alpha s1-kappa-Cn, beta-kappa-Cn and alpha s2-kappa-Cn, was observed in the progeny of double heterozygous rams. The casein genes are thus clustered in the ovine species as in the case of other mammals.     

Molecular characterization of the porcine MHC class I region

A porcine cosmid library was screened with a human MHC class I cDNA. Four positive clones were isolated and mapped with different restriction endonucleases. Altogether nine SLA class I genes were identified and their positions located within restriction maps. Sizes of class I homologous DNA sequences varied between 3600 and 5800bp. The distances between these regions ranged from 11900 to 22200bp.     

Restriction maps and restriction fragment length polymorphisms of the human 21-hydroxylase genes

Restriction maps were constructed for the two human 21-hydroxylase genes (21-OHA and 21-OHB) by using DNA from subjects homozygous for a deletion of each gene. Comparing the patterns of these two genes, a KpnI restriction site occurred in the 21-OHA gene in place of a TaqI site in the 21-OHB gene about 1-kb from the 5' end of the gene, and an extra EcoRI site was located 500 bp 5' to the common EcoRI site. The DNA of fourteen unrelated normal subjects was digested with nine restriction endonucleases (AccI, BamHI, BgIII, EcoRI, HindIII, KpnI, MspI, SacI and TaqI). Restriction fragment length polymorphisms were found with EcoRI, HindIII and AccI that resulted from polymorphic endonuclease sites outside the genes.     

There are approximately 20 actin gene in the human genome.

By three different lines of evidence there are approximately 20 copies of actin genes in the human genome. Firstly, the rate of hybridisation of a mouse actin probe to human DNA indicates that there are a minimum of 20 complementary copies of the actin sequence per genome. Secondly, this probe hybridises to 17-20 bands in Southern blots of restriction enzyme digests of total human DNA. Most of these bands hybridise with both 3' and 5' fragments of the cDNA and are therefore likely to contain the entire gene sequence. Thirdly, we have picked 12 actin recombinants from a genomic library, and at the level of restriction enzymes mapping these represent nine different genes. Probability calculations indicate that these recombinants were picked from a pool of at least 20 different genes.     

Restriction endonuclease activities in the legionellae

Fifteen legionellae isolates were studied for the presence of restriction endonucleases. At least three different specificities were found in nine of these isolates. One particular activity was present in two groups of isolates isolated from widely separated geographic areas.     

Multiplicity of deoxyribonucleic acid sequences with homology to a cloned complementary deoxyribonucleic acid coding for rat phenobarbital-inducible cytochrome P-450

We previously identified cDNA clones for rat cytochrome P-450 of the phenobarbital-inducible type by sequence analysis [Fujii-Kuriyama, Y., Mizukami, Y., Kawajiri, K., Sogawa, K., & Muramatsu, M. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2793-2797]. With these cloned cDNAs as probe, the multiplicity of phenobarbital-inducible cytochrome P-450 gene in rat genome was investigated by three approaches. The first approach was the Cot analysis of the total rat liver DNA under conditions of DNA excess. With internal and external markers used as gene-number standards, the reassociation kinetics were studied, which suggested the presence of approximately six genes or gene-like sequences hybridizable to phenobarbital-inducible cytochrome P-450 cDNA per rat haploid genome. The second was the isolation of the cytochrome P-450 genes from a rat genomic library. From a screening of about 1 X 10(6) plaques, nine clones with an approximately 15-kb insert were isolated. Restriction maps and Southern blot analysis of the cloned DNAs showed that six out of nine isolated clones contained DNA inserts independent of one another. The third was Southern blot analysis of rat genomic DNA with restriction enzyme EcoRI. Approximately 12 positive bands were demonstrated with the cDNA probe, seven to eight of which showed the same mobilities as the fragments in the isolated six genomic clones, suggesting that some other genes or gene-like DNA sequences remained to be cloned.     

Mytilus edulis Histone Gene Clusters Containing Only H1 Genes

We isolated five different phage clones containing histone gene clusters with up to five H1 genes per phage clone from a Mytilus edulis genomic library. Among these H1 genes, nine gene types coding for five different H1 proteins have been identified. All H1 histone genes were located on repetitive restriction fragments with only slightly different sizes. The H1 coding regions show highly related sequences, suggesting that the multitude of H1 genes has evolved by gene duplication events. Core histone genes could not be found on these five Mytilus edulis genome fragments. Received: 28 July 1998 / Accepted: 17 May 1999     

tRNA genes in mycobacteria: organization and molecular cloning.

DNAs from nine mycobacteria cleaved with restriction endonucleases were hybridized with cDNA probes synthesized to tRNAs from Mycobacterium tuberculosis and Mycobacterium smegmatis. The tRNA genes are conserved, but their gross genomic organization has diverged in six of the nine species examined. Organisms of the M. tuberculosis H37Ra and H37Rv-M. bovis BCG complex appeared to have identical tRNA gene organization and were indistinguishable from each other. M. tuberculosis and M. smegmatis tRNA-derived cDNA probes hybridized differentially to tRNA-coding DNA segments in five of the species examined, suggesting the existence of qualitatively different tRNA pools in these slow- and fast-growing species. Mycobacterial DNAs hybridized with cDNA synthesized to 23S plus 16S rRNAs from Escherichia coli, and the data suggested that the tRNA genes map close to the rRNA genes. A gene bank of M. tuberculosis H37Rv DNA was constructed, and a recombinant plasmid, pSB2, coding for tRNA(s) and rRNA(s) was partially characterized. Plasmid pSB2 recognized a SalI restriction fragment length polymorphism (RFLP) in M. tuberculosis H37Rv and H37Ra; however, the RFLP is not linked to the tRNA-coding region. To the best of our knowledge, this is the first report of an RFLP which distinguishes the pathogenic strain M. tuberculosis H37Rv from its avirulent derivative H37Ra.     

Functional characterization of human immunodeficiency virus type 1 nef genes in patients with divergent rates of disease progression.

We have studied the sequence and function of the human immunodeficiency virus type 1 (HIV-1) nef genes from nine patients with highly divergent rates of disease progression enrolled in a longitudinal study of HIV disease. Over an average of 7.8 years of follow-up, three patients had net positive changes in CD4+ T-cell counts, three patients had net negative changes in CD4+ T cells but did not develop AIDS, and three patients progressed to AIDS. The nef gene from each of these patients was amplified and cloned, and the sequence of 8 to 10 clones was determined. Only 2 of 88 (2.3%) nef genes recovered from these nine patients were grossly defective. Moreover, there was no relationship between the phylogeny of nef sequences and the corresponding rates of disease progression from these patients. Representative nef genes from all nine patients were tested for their abilities to downregulate cell surface CD4 in a transient-transfection assay. There was no correlation found between the functions of the nef genes from these patients and their corresponding rates of disease progression. We conclude that the nef gene is not a common mediator of the rate of HIV disease progression in natural infection.     

Ras-like genes and gene families in the mouse

Four humanRAS-like cDNAs and a mouse genomic DNA fragment were used to define novel mouseRas-like genes and gene families. Inheritance of DNA restriction fragment length variants associated with these genes in recombinant inbred and backcross mice allowed definition of 12 genetic loci, nine of which were mapped, to chromosomes (Chr) 2, 4, 7, 8, 9, and 17. Two possible clusters ofRas-like and/or G protein genes were identified, on Chrs 9 and 17.     

Mutations with Dominant Effects on the Behavior and Morphology of the Nematode CAENORHABDITIS ELEGANS

We have analyzed 31 mutations that have dominant effects on the behavior or morphology of the nematode Caenorhabditis elegans. These mutations appear to define 15 genes. We have studied ten of these genes in some detail and have been led to two notable conclusions. First, loss of gene function for four of these ten genes results in a wild-type phenotype; if these genes represent a random sample from the genome, then we would estimate that null mutations in about half of the genes in C. elegans would result in a nonmutant phenotype. Second, the dominant effects of mutations in nine of these ten genes are caused by novel gene functions, and in all nine cases the novel function is antagonized by the wild-type function.     

Up-regulation of stearoyl-CoA desaturase 1 and elongase 6 genes expression in rat lipogenic tissues by chronic food restriction and chronic food restriction/refeeding

Successful treatment of obesity and related diseases by chronic food restriction requires the understanding of the effect of such nutritional therapy on the expression of genes which have been implicated to be involved in some diseases associated with obesity. The purpose of this study was to examine the effect of chronic food restriction and chronic food restriction/refeeding on lipogenic enzymes, especially the expression of genes encoding the stearoyl-CoA desaturase 1 (Scd1) and elongase6 (Elovl6) in rat liver and adipose tissue. We found that both chronic food restriction and chronic food restriction/refeeding caused increased expression of the Scd1 and Elovl6 genes in both the liver and adipose tissue. The increase was more pronounced in case of chronic food restriction/refeeding (several-fold increase) than that in chronic food restriction alone (two to threefold increase). Essentially, similar results were obtained when the expression of fatty acid synthase, acetyl-CoA carboxylase, ATP-citrate lyase, and malic enzyme genes was studied. Moreover, we found that chronic food restriction and short-term fasting exert opposite effects on the expression of lipogenic enzymes genes. The increased expression of the genes encoding Scd1, Elovl6, and other key lipogenic enzymes may favor fat storage after chronic food restriction/refeeding and may be part of the molecular mechanism by which food restriction/refeeding increases body weight and enhances susceptibility to insulin resistance.     

Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes.

We constructed nine sets of oligonucleotide primers on the basis of the results of DNA hybridization of cloned genes from Neurospora crassa and Aspergillus nidulans to the genomes of select filamentous ascomycetes and deuteromycetes (with filamentous ascomycete affiliations). Nine sets of primers were designed to amplify segments of DNA that span one or more introns in conserved genes. PCR DNA amplification with the nine primer sets with genomic DNA from ascomycetes, deuteromycetes, basidiomycetes, and plants revealed that five of the primer sets amplified a product only from DNA of the filamentous ascomycetes and deuteromycetes. The five primer sets were constructed from the N. crassa genes for histone 3, histone 4, beta-tubulin, and the plasma membrane ATPase. With these five primer sets, polymorphisms were observed in both the size of and restriction enzyme sites in the amplified products from the filamentous ascomycetes. The primer sets described here may provide useful tools for phylogenetic studies and genome analyses in filamentous ascomycetes and deuteromycetes (with ascomycete affiliations), as well as for the rapid differentiation of fungal species by PCR.     

Identification and location of nine T5 bacteriophage tRNA genes by DNA sequence analysis.

Sequence analysis of two DNA fragments generated from bacteriophage T5 DNA by restriction with Hpa I and Hae III has resulted in the detection and localization of nine tRNA genes (His, two Ser genes, Leu, Val, Lys, fMet, Pro, and Ile). The genes which code for tRNAs His and Leu are partials, whereas the remaining genes are complete. A majority of the tRNA genes are located in close proximity to one another. A unique feature of the Pro and Ile genes is that their DNA sequence overlap.     

Genes encoding the alpha and beta chains of follicle-stimulating hormone are not sites for the Booroola (FecB) mutation in sheep.

Bovine cDNA probes for the beta-subunit of follicle-stimulating hormone beta (FSH beta) and the alpha-subunit of the glycoprotein hormones identify genetic variation (polymorphic restriction fragments) near these genes in sheep. The inheritance of the polymorphic restriction fragments was studied in half-sibling pedigrees generated by mating heterozygous (B+) rams to non-carrier (++) ewes so that the co-inheritance or genetic linkage to the Booroola (FecB) locus and the alpha- and beta-subunits of FSH could be analysed. Genetic recombination was observed between the FSH beta locus and the FecB locus in all five families studied and between the alpha-subunit and the FecB locus in the two families studied. We conclude that the FecB mutation does not lie within the FSH beta- or alpha-subunit genes encoding the heterodimeric hormone FSH, and that the high concentrations of FSH observed in carrier ewes must result from indirect actions of the FecB mutation on the synthesis, processing, storage, release or metabolism of FSH.     

Organization and function of the mcrBC genes of Escherichia coli K-12

Many natural DNA sequences are restricted in Escherichia coli K-12, not only by the classic Type I restriction system EcoK, but also by one of three modification-specific restriction systems found in K-12. The McrBC system is the best studied of these. We infer from the base composition of the mcrBC genes that they were imported from an evolutionarily distant source. The genes are located in a hypervariable cluster of restriction genes that may play a significant role in generation of species identity in enteric bacteria. Restriction activity requires the products of two genes for activity both in vivo and in vitro. The mcrB gene elaborates two protein products, only one of which is required for activity in vitro, but both of which contain a conserved amino acid sequence motif identified as a possible GTP-binding site. The mcrC gene product contains a leucine heptad repeat that could play a role in protein-protein interactions. McrBC activity in vivo and in vitro depends on the presence of modified cytosine in a specific sequence context; three different modifications are recognized. The in vitro activity of this novel multi-subunit restriction enzyme displays an absolute requirement for GTP as a cofactor.     

First trimester prenatal diagnosis and detection of carriers of haemophilia A using the linked DNA probe DX13.

Although the use of a gene specific deoxyribonucleic acid (DNA) probe is the method of choice for detecting carriers of genes for rare genetic disorders, there will always be families in which such probes cannot be used because key subjects are not informative for restriction fragment length polymorphisms in or around the gene. In these cases closely linked DNA markers have to be used. An X chromosome specific DNA probe, DX13, which is closely linked to the haemophilia A locus on the X chromosome, was used for early prenatal diagnosis in two cases and to detect carriers in a series of nine possible heterozygote women. The first reported crossover between DX13 and the factor VIII:C locus was observed in this study. There are complexities inherent in using any linked DNA probe for assignment of genes, but such techniques are clinically important.     

Molecular analysis of sorghum resistance to the greenbug (Homoptera: Aphididae)

Chromosomal regions of sorghum, Sorghum bicolor (L.) Moench, conferring resistance to greenbug, Schizaphis graminum (Rondani), biotypes C, E, I, and K from four resistance sources were evaluated by restriction fragment-length polymorphism (RFLP) analysis. At least nine loci, dispersed on eight linkage groups, were implicated in affecting sorghum resistance to greenbug. The nine loci were named according to the genus of the host plant (Sorghum) and greenbug (Schizaphis graminum). Most resistance loci were additive or incompletely dominant. Several digenic interactions were identified, and in each case, these nonadditive interactions accounted for a greater portion of the resistance phenotype than did independently acting loci. One locus in three of the four sorghum crosses appeared responsible for a large portion of resistance to greenbug biotypes C and E. None of the loci identified were effective against all biotypes studied. Correspondingly, the RFLP results indicated resistance from disparate sorghums may be a consequence of allelic variation at particular loci. To prove this, it will be necessary to fine map and clone genes for resistance to greenbug from various sorghum sources.     

Cloning and characterization of the ribosomal genes of the sea-urchin Paracentrotus lividus. Heterogeneity of the multigene family

A Paracentrotus lividus genomic library was constructed using sperm DNA prepared from a single animal. The DNA was fragmented by partial digestion with DNase II, sized on a preparative agarose gel and inserted in the Pst I site of pBR 322 by the dG X dC tailing method. Recombinant plasmids containing ribosomal DNA were isolated, a restriction map of the gene was determined and the 18S and 26S transcribed sequences were located by S1 protection mapping. The organization of the ribosomal genes in genomic DNA of individual animals and of a pool of animals was studied by blot-hybridization of the restriction fragments, using as probes nick-translated 32P-labelled cloned ribosomal DNA fragments or 18S and 26S sea-urchin ribosomal RNA. The repeat length of the ribosomal unit was about 10.5 X 10(3) bases. A comparison of the restriction patterns of DNA from different animals showed a marked sequence heterogeneity in the spacer region of these genes. Variations of about 200 base pairs were detectable in the length of the spacer of some individuals.