R-ISSR as a new tool for genomic fingerprinting, mapping, and gene tagging

In the present study we propose and test the concept of R-ISSR, a new tool for genomic fingerprinting, mapping, and gene tagging. The concept is based on the fact that primers for inter-simple sequence repeat (ISSR) and random-amplified polymorphic DNA (RAPD) analysis elicit different genomic information, and the combined use of these 2 kinds of primers in the same polymerase chain reaction (PCR) reactions might reveal new genomic loci that could not be detected with either technique alone. The feasibility of this tool was first electronically simulated with sequence analysis software andArabidopsis chromosome sequence. Next, different combinations of ISSR and RAPD primers were applied in real PCR reactions to detect new genomic loci in 2 maize lines (Q319 and 1145). Sequencing gels were used to separate PCR products and showed good resolving ability in comparison with agarose gels. RAPD primers could be successfully used with ISSR primers for the detection of new genomic loci and applied in a new way for genomic mapping, fingerprinting, and gene tagging.     

Human thymidylate synthase gene: isolation of phage clones which cover a functionally active gene and structural analysis of the region upstream from the translation initiation codon

Two genomic DNA fragments partially encoding human thymidylate synthase (TS) [EC 2.1.1.45] were previously cloned in lambda phage from the mouse cell transformant, but had no transforming activity on mouse TS-negative mutant cells. In this study, an additional genomic DNA for human TS was cloned and demonstrated to have the transforming activity in combination with one of the two previously cloned DNAs and to produce human TS mRNA. The two transforming genomic DNAs overlapped and covered a region of 23 kb in total. Using fragments from one of these DNAs, the structure of the 1.2-kb region around the ATG initiator codon of the TS gene was analyzed in relation to regulatory sequences of the gene. Sequence determination demonstrated the presence of an unusual inverted repeat consisting of a triple tandem repeat of a 28-bp sequence and an inverted sequence of the same length. These sequences can form three possible, stable, stem-loop structures, which may be interconvertible. Based on S1 nuclease mapping data and a line of circumstantial evidence, we deduced two major mRNA cap sites within the inverted sequence. Comparison of the human and mouse sequences upstream from the ATG initiator codon revealed many significant blocks of sequence homology, especially in the regions around the deduced cap sites.     

A novel homologue of the TIAP/m-survivin gene

The inhibitor of apoptosis (IAP) proteins comprise a highly conserved gene family that prevents cell death in response to a variety of stimuli. TIAP/m-survivin, a murine homologue of human Survivin, is a member of the IAP family. TIAP/m-survivin has one baculovirus IAP repeat (BIR) and lacks a C-terminal RING finger motif. Here we identified the genomic DNA region (TIAP-2) that is homologous to the TIAP/m-survivin gene by a low stringency genomic DNA hybridization. The region is on the chromomsome 9 which is distinct from that (chromosome 11) of the TIAP/m-survivin gene, and contains DNA sequence similar to a part of the BIR and the 3' side of the TIAP/m-survivin gene and the sequence homology between them is 92%. Expression of TIAP-2 mRNA was detected in various murine tissues by RT-PCR. Although expression of TIAP/m-survivin mRNA is upregulated in synchronized cells at S to G2/M phase of the cell cycle, expression of TIAP-2 mRNA was constant in the cell cycle, suggesting the different role of TIAP-2 from that of TIAP/m-survivin.     

Isolation and deduced amino acid sequence of the gene encoding gp115, a yeast glycophospholipid-anchored protein containing a serine-rich region

gp115 is a N- and O-glycosylated protein of Saccharomyces cerevisiae. It is also modified by addition of glycosylphosphatidylinositol, which anchors the protein to the plasma membrane. The gene encoding gp115 (GGP1) has been cloned by a two-step procedure. By an immunoscreening of a yeast genomic DNA library in the expression vector lambda gt11, a 3'-terminal 0.9-kilobase portion of the gene has been isolated and then used as a molecular probe to screen a yeast genomic DNA library in YEp24. In this way, the whole GGP1 gene has been cloned. Its identity with the gp115 gene has been confirmed by gene disruption, which has also indicated that the function of gp115 is not essential for cell viability. The features of the sequence are also entirely consistent with it corresponding to the gp115 gene. The nucleotide sequence of GGP1 predicts a 60-kDa polypeptide, in agreement with the molecular mass of the gp115 precursor detected in sec53 mutant cells at restrictive temperature. Two hydrophobic sequences, one NH2- and the other COOH-terminal were found. The former has the features of the cleavable signal sequence, which allows the entry of proteins in the secretory pathway. The latter could be the signal sequence that has to be removed during the addition of glycosylphosphatidylinositol. The predicted amino acid sequence of gp115 shows 10 sequons for N-glycosylation and a high proportion of serine-threonine residues (22%) that could provide several sites for O-glycosylation. The unusual concentration of 27 serines in the COOH-terminal portion of the protein shares homology with a similar polyserine repeat of the serine repeat antigen (SERA protein) of Plasmodium falciparum. A two-dimensional analysis of the "in vitro" translational product of the GGP1 mRNA has been carried out, allowing the identification of the "in vivo" gp115 precursor in a two-dimensional gel.     

Genomic organization and context of a trypanosome variant surface glycoprotein gene family.

We have defined the genomic organization and genomic context of a Trypanosoma brucei brucei gene family encoding variant surface glycoproteins (VSGs). This gene family is neither tandemly repeated nor closely linked in the genome, and is not located on small or intermediate size chromosomes. Two dispersed repeated sequence elements, RIME-ingi and the upstream repeat sequence, are linked to members of this gene family; however, the upstream repeat sequences are closely linked only to the basic copy. In other isolates of T.b. brucei this gene family appears conserved with some variation; a restriction fragment length polymorphism found among these isolates suggests the hypothesis that VSG genes may occasionally be diploid. A model accounting for both the generation of dispersed families of VSG genes, and for the interstrain variability of VSG genes, is proposed.     

A virus-inducible tobacco gene encoding a glycine-rich protein shares putative regulatory elements with the ribulose bisphosphate carboxylase small subunit gene

cDNA to an mRNA that is strongly induced in Samsun NN tobacco after tobacco mosaic virus (TMV) infection or salicylic acid treatment was used to probe a genomic blot and to screen a genomic library. The mRNA corresponds to a family of approximately eight genes, four of which were cloned. The sequence of the genes and flanking DNA in two clones was determined. One gene was found to contain an intron of 555 bp; S1-nuclease mapping studies indicated that this gene is expressed. The other gene is interrupted by an intron of 1,954 bp and is probably not expressed after TMV infection. The genes encode a protein of 109 amino acids with a putative N-terminal signal peptide of 26 amino acids. The protein contains a high proportion of glycine (25%) and charged amino acids (29%), suggesting that it may be a cell wall component. A comparison of the upstream sequences of the genes encoding the glycine-rich protein and the pathogenesis-related protein 1a showed only limited homology, although both genes are TMV- and salicylic acid-inducible. However, the upstream sequence of the glycine-rich protein gene contains a 64-bp inverted repeat that occurs in a similar position in the tobacco ribulose bisphosphate carboxylase small subunit gene.     

Rapid detection of CA polymorphisms in cloned DNA: application to the 5'' region of the dystrophin gene.

To identify CA repeats in genomic sequences which had been previously subcloned into plasmids, we performed PCR using a (CA)n primer and a flanking vector primer on the genomic inserts. By incorporation of a restriction enzyme site into the (CA)n primer, we have been able to subclone the genomic DNA so that the sequence flanking the CA repeat is readily determined. Primers can then be designed to amplify across the CA repeat in patient DNA samples. Application of this technique to genomic DNAs surrounding the upstream "brain" promoter of the dystrophin gene has led to the discovery of four new CA repeats. Three of these repeats are highly polymorphic, with PICs ranging from .586 to .768. The location of these markers at the extreme 5' terminus of the dystrophin gene, together with their high degree of polymorphism and ease of assay, makes them ideal for linkage analysis in families with Duchenne muscular dystrophy.     

UbC gene allele frequency in Korean population and novel UbC mosaic repeat unit formation

The genomic structural organization of human UbC CDS repeat units could be representative of concerted evolution. The structure of the UbC gene and its repeat unit number frequency at scales of different human ethnic populations remain to be sufficiently determined. In this study, we performed comparative analysis of UbC CDS regions in genomes from 140 Korean individuals. We found that the UbC gene allele types 9, 8 and 7 are present in the Korean population in proportions of 97.1%, 0.4% and 2.5%, respectively. Interestingly, we discovered that the allele types 7 and 8 harbor the novel UbC gene mosaic repeat units 3??5 (combined between sequence parts derived from standard repeat units 3 and 5) and 8??9 (combined between sequence parts derived from standard repeat units 8 and 9) within their sequence structures, respectively. Our analysis showed that the novel mosaic repeat unit 3^5 lacks the highly human-specific amino acid S38, implying a functional consequence. These results suggest that the genomic organization of UbC repeat units is still undergoing dynamic structural changes due to concerted evolution through unequal crossing-over. Our results could represent valuable data for future investigations related to treating genetic diseases caused by UbC gene mutations and variations.     

The human loricrin gene.

Loricrin is the major protein component of the cornified cell envelope of terminally differentiated mammalian epidermal (stratum corneum) cells. Using a specific human cDNA clone, we have isolated and characterized the human loricrin gene. We show that it has a very simple structure of a single intron of 1188 base pairs (bp) in the 5'-untranslated region; there are no introns in coding sequences. By use of rodent-human somatic cell hybrids, followed by in situ hybridization with a biotin-labeled genomic DNA clone, the single-copy gene maps to chromosome location 1q21. Polymerase chain reaction analyses of genomic DNAs from different individuals show that human loricrin consists of two allelic size variants, due to sequence variations in its second glycine loop domain, and these variants segregate in the human population by normal Mendelian mechanisms. Furthermore, there are multiple sequence variants within these two size class alleles due to various deletions of 12 bp (4 amino acids) in the major loop of this glycine loop domain. By use of a specific loricrin antibody, we show by immunogold electron microscopy that loricrin initially appears in the granular layer of human epidermis and forms composite keratohyalin granules with profilaggrin, but localizes to the cell periphery (cell envelope) of fully differentiated stratum corneum cells.     

Detection of px gene product of bovine leukemia virus in infected cells

Bovine leukemia virus (BLV), like its closest relatives human T-cell leukemia virus-I and II, contain a 'px' gene, between the 'env' gene and the 3' long terminal repeat in its genome. A monoclonal antibody prepared against a synthetic oligopeptide whose sequence was deduced from highly conserved region of 'px' gene of BLV, was used to detect the presence of 'px' gene product in chronically BLV infected synchronised cells. By immunoperoxidase staining the 'px' gene product was detected maximum after 6-9 hr after synchronization in the nucleus of the cells which demonstrated the close interaction of it with viral DNA which is integrated with host cell genome.     

Nuclear proteins preferentially bound to the beta-globin gene: cellular specificity and variation during terminal differentiation of mouse erythroblasts

Restriction fragments of the mouse beta major globin gene and of the long terminal repeat (LTR) DNA fragment of the mouse mammary tumor provirus as a control, were used to analyze the specificity of DNA-protein interactions in nuclear extracts of mouse erythroleukemia (MEL) cells and of other differentiated mouse cultured cell lines. After gel electrophoresis and transfer to nitrocellulose, DNA-binding proteins with a preferential affinity for the cloned beta-globin genomic sequence were characterized and related to the level of globin gene expression during induction of differentiating mouse erythroblasts. Two proteins (110 K and 75 K) appear in differentiated MEL cells while another one (100 K), for which we have localized the binding site on the beta-globin gene, is present only in immature MEL cells.     

Isolation of three novel Drosophila melanogaster genes containing repetitive sequences rich in GCN triplets.

Several cDNA and genomic clones were isolated from Drosophila melanogaster gene libraries by hybridization with a region of a mammalian gene that contains a simple repetitive sequence of six GCN repeats. One of the cDNA clones, E6, was completely sequenced and it was shown that it contains a region of 16 GCN repeats; these repeats encode a polyalanine stretch within a long open reading frame. The sequencing of three different genomic clones (A, B, and D) revealed that all the isolated Drosophila clones are similar to one another in a short region containing variable numbers of the GCN repeat. The genomic clone B was found to be the genomic counterpart of the cDNA clone E6. The other genomic clones, A and D, also hybridize with Drosophila cDNA clones at high stringency. These results indicate that the short GCN repetitive sequences, which we have named ala, are found within transcribed regions of the Drosophila genome. These Drosophila genes containing the ala repeat do not show significant sequence similarity to any presently known gene; we have named these novel genes ala-A, ala-B, and ala-D. The cDNA clone from gene ala-B was named ala-E6.