Showing papers on "genomic DNA published in 1986"

Analysis of enzymatically amplified beta-globin and HLA-DQ alpha DNA with allele-specific oligonucleotide probes.

Abstract: Allelic sequence variation has been analysed by synthetic oligonucleotide hybridization probes which can detect single base substitutions in human genomic DNA. An allele-specific oligonucleotide (ASO) will only anneal to sequences that match it perfectly, a single mismatch being sufficient to prevent hybridization under appropriate conditions. To improve the sensitivity, specificity and simplicity of this approach, we used the polymerase chain reaction (PCR) procedure to enzymatically amplify a specific segment of the beta-globin or HLA-DQ alpha gene in human genomic DNA before hybridization with ASOs. This in vitro amplification method, which produces a greater than 10(5)-fold increase in the amount of target sequence, permits the analysis of allelic variation with as little as 1 ng of genomic DNA and the use of a simple 'dot blot' for probe hybridization. As a further simplification, PCR amplification has been performed directly on crude cell lysates, eliminating the need for DNA purification.

Human basic fibroblast growth factor: nucleotide sequence and genomic organization.

Abstract: Clones encoding the angiogenic endothelial cell mitogen, basic fibroblast growth factor (FGF), have been isolated from human cDNA libraries made from kidney, fetal heart, fetal liver, term placenta, and a breast carcinoma. Basic FGF cDNA clones are present in these libraries at very low levels when compared to the quantity of the growth factor in the tissues. This observation, combined with the fact that several of the clones represent unspliced transcripts, suggests that cytoplasmic basic FGF mRNA is unstable and that the protein is stored in tissues. The amino acid sequence of human basic FGF, deduced from the sequence of these cDNAs and from genomic clones, is 99% homologous to that of bovine basic FGF, implying a strong selection pressure for maintenance of function and structure. As with the bovine factor, human basic FGF does not appear to have a signal peptide sequence. Southern blot analysis of human genomic DNA and mapping of the cloned gene shows that there is only one basic FGF gene. All of the basic, heparin-binding endothelial cell mitogens of similar amino acid composition that have been described must therefore be products of this single gene.

Identification of a nonhistone chromosomal protein associated with heterochromatin in Drosophila melanogaster and its gene.

Abstract: Monoclonal antibodies were prepared against a fraction of nuclear proteins of Drosophila melanogaster identified as tightly binding to DNA. Four of these antibodies were directed against a 19-kilodalton nuclear protein; immunofluorescence staining of the polytene chromosomes localized the antigen to the alpha, beta, and intercalary heterochromatic regions. Screening of a lambda gt11 cDNA expression library with one of the monoclonal antibodies identified a recombinant DNA phage clone that produced a fusion protein immunologically similar to the heterochromatin-associated protein. Polyclonal sera directed against the bacterial lacZ fusion protein recognized the same nuclear protein on Western blots. A full-length cDNA clone was isolated from a lambda gt10 library, and its DNA sequence was obtained. Analysis of the open reading frame revealed an 18,101-dalton protein encoded by this cDNA. Two overlapping genomic DNA clones were isolated from a Charon 4 library of D. melanogaster with the cDNA clone, and a restriction map was obtained. In situ hybridization with these probes indicated that the gene maps to a single chromosome location at 29A on the 2L chromosome. This general strategy should be effective for cloning the genes and identifying the genetic loci of chromosomal proteins which cannot be readily assayed by other means.

Construction of genetic linkage maps in maize and tomato using restriction fragment length polymorphisms.

Abstract: Genetic linkage maps were constructed for both maize and tomato, utilizing restriction fragment length polymorphisms (RFLPs) as the source of genetic markers In order to detect these RFLPs, unique DNA sequence clones were prepared from either maize or tomato tissue and hybridized to Southern blots containing restriction enzyme-digested genomic DNA from different homozygous lines A subsequent comparison of the RFLP inheritance patterns in F2 populations from tomato and maize permitted arrangement of the loci detected by these clones into genetic linkage groups for both species

Ethylene-regulated gene expression: molecular cloning of the genes encoding an endochitinase from Phaseolus vulgaris.

Abstract: A full-length copy of bean leaf chitinase mRNA has been cloned. The 1146-base-pair insert of pCH18 encodes the 27-residue amino-terminal signal peptide of the precursor and 301 residues of the mature protein. Utilizing pCH18 as a hybridization probe, we have shown that the increase in translatable chitinase mRNA seen upon ethylene treatment of bean seedlings is due to a 75- to 100-fold increase in steady-state mRNA levels. Southern blot analysis of bean genomic DNA revealed that chitinase is encoded by a small, multigene family consisting of approximately four members. From our nucleotide sequence analysis of five additional chitinase cDNA clones, it appears that at least two of these genes are expressed. Three of the bean chitinase genes have been isolated from a Sau3A genomic library and partially characterized.

The PEP4 gene encodes an aspartyl protease implicated in the posttranslational regulation of Saccharomyces cerevisiae vacuolar hydrolases.

Abstract: pep4 mutants of Saccharomyces cerevisiae accumulate inactive precursors of vacuolar hydrolases. The PEP4 gene was isolated from a genomic DNA library by complementation of the pep4-3 mutation. Deletion analysis localized the complementing activity to a 1.5-kilobase pair EcoRI-XhoI restriction enzyme fragment. This fragment was used to identify an 1,800-nucleotide mRNA capable of directing the synthesis of a 44,000-dalton polypeptide. Southern blot analysis of yeast genomic DNA showed that the PEP4 gene is unique; however, several related sequences exist in yeasts. Tetrad analysis and mitotic recombination experiments localized the PEP4 gene proximal to GAL4 on chromosome XVI. Analysis of the DNA sequence indicated that PEP4 encodes a polypeptide with extensive homology to the aspartyl protease family. A comparison of the PEP4 predicted amino acid sequence with the yeast protease A protein sequence revealed that the two genes are, in fact, identical (see also Ammerer et al., Mol. Cell. Biol. 6:2490-2499, 1986). Based on our observations, we propose a model whereby inactive precursor molecules produced from the PEP4 gene self-activate within the yeast vacuole and subsequently activate other vacuolar hydrolases.

Isolation of cDNA clones coding for the catalytic subunit of mouse cAMP-dependent protein kinase

Abstract: mRNA coding for the catalytic (C) subunit of cAMP-dependent protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) was partially purified from bovine testis by polysome immunoadsorption and oligo(dT)-chromatography. This enriched mRNA preparation was used to prepare and differentially screen a cDNA library. One of the selected cDNA clones was shown to hybrid-select mRNA coding for a 40-kDa protein that was specifically precipitated with antibodies to the C subunit. This bovine cDNA clone was then used to isolate a series of mouse cDNA clones that are complementary to the entire mouse C subunit mRNA. The mouse clones code for a protein of 351 amino acids that shows 98% homology to the bovine C subunit and hybridize to a single mRNA of 2.4 kilobases in mouse heart and brain. Southern blot analysis of total genomic DNA suggests that there is a single mouse gene coding for the C subunit. mRNA levels for both the C subunit and the type I regulatory subunit in various mouse tissues and cell lines were quantitated and compared by using single-stranded RNA probes prepared with SP6 polymerase.

Genomic analysis of the human B-lymphotropic virus (HBLV).

Abstract: The human B-lymphotropic virus (HBLV) has a double-stranded DNA genome of greater than 110 kilobase pairs, which is consistent with its morphological classification as a herpesvirus. A 9000-base pair cloned probe of HBLV detected specific sequences in DNA and RNA of infected cells but did not hybridize to the genomic DNA of other human herpesviruses including the Epstein-Barr virus, human cytomegalovirus, herpes simplex type I, and varicella-zoster virus. Conversely, while probes obtained from each of the known human herpesvirus readily detected the homologous viral DNA, they did not hybridize to genomic HBLV DNA. This evidence, in addition to serological and morphological distinctions and the biological effects of this virus demonstrate that HBLV is a novel human herpesvirus.

Rapid in Vivo Effects of Glucocorticoids on the Integrity of Rat Lymphocyte Genomic Deoxyribonucleic Acid

Abstract: Glucocorticoid-induced lymphocytolysis has been studied for many years; however, the mechanism of lymphoid cell death has not been elucidated. In this study we have investigated the effects of glucocorticoids on the lymphocyte genome using the rat thymocyte model. Adrenalectomized rats were injected ip with dexamethasone (DEX) and killed thereafter. The thymus gland was removed, and DNA was extracted from isolated thymocytes and then separated electrophoretically on 1.8% agarose gels. Administration of glucocorticoids in vivo resulted in the cleavage of lymphocyte DNA at internucleosomal intervals. Genomic DNA separated on agarose gels from DEX-treated rats appeared as a ladder of DNA fragments which were multiples of about 180 base pairs, while DNA from control rats appeared as a single high mol wt band. This cleavage of thymocyte DNA was a rapid effect of adrenal steroid treatment and occurred before cell death. Thymocyte DNA fragmentation increased with time after DEX treatment and the dose of half-maximal response in vivo was estimated to be about 1.8 X 10(-8) M. Internucleosomal cleavage of DNA was only observed in lymphoid tissues (thymus and spleen), but not in other glucocorticoid-sensitive tissues (kidney, liver, heart, brain, or testis). Treatment of rats with estrogen, androgen, or progestin failed to elicit thymocyte DNA degradation. Glucocorticoid-induced DNA cleavage was partly inhibited by the glucocorticoid antagonist RU 486 (17 beta-hydroxy-11 beta,4-dimethylaminophenyl-17 alpha-propynl-estra-4,9-diene-3-one). These findings suggest that glucocorticoids activate, via a receptor-mediated process, an endonuclease-like activity in lymphoid tissues which cleaves the lymphocyte genome at internucleosomal sites. Activation of this nuclease by glucocorticoids precedes lymphocytolysis and may represent the hormonal regulation of programmed cell death.

Pancreatic phospholipase A2: isolation of the human gene and cDNAs from porcine pancreas and human lung.

Abstract: Oligonucleotide probes synthesized using the published protein sequence for porcine pancreatic phospholipase A2 (PLA2; Puijk et al., 1977), were used to screen a cDNA library made from porcine pancreas. One resulting clone which encoded the entire porcine PLA2 enzyme was then used to screen various human cDNA and gene libraries. A 562-bp cDNA clone from human lung and two overlapping genomic clones encoding the identical transcript were obtained. These clones encoded a 126-residue peptide corresponding to human "pancreatic" PLA2. The gene spanned 4.9 kb and contained three introns of 1692, 800, and about 2650 bases, respectively. Hybridization of the human cDNA against blots of total human DNA detected the same set of fragments contained in both genomic clones and failed to detect more than one distinct gene. Under equivalent conditions, the same probe detected two discrete bands in cobra (Naja naja) genomic DNA. Apparently, there is a single or small number of identical human genes encoding "pancreatic" ...

Isolation and characterization of DNA sequences amplified in multidrug-resistant hamster cells

Abstract: The mechanism by which mammalian cells acquire resistance to chemotherapeutic agents has been investigated by using molecular genetic techniques. LZ and C5, two independently derived multidrug-resistant Chinese hamster cell lines, share specific amplified DNA sequences. We demonstrate that commonly amplified DNA sequences reside in a contiguous domain of approximately equal to 120 kilobases (kb). We report the isolation of this DNA domain in cosmid clones and show that the level of amplification of the domain is correlated with the level of resistance in multidrug-resistant cell lines. The organization of the amplified domain was deduced by a unique approach utilizing in-gel hybridization of cloned DNA with amplified genomic DNA. We show that the entire cloned region is amplified in adriamycin-resistant LZ cells and independently derived, colchicine-resistant C5 cells. A mRNA species of approximately equal to 5 kb is encoded by a gene located within the boundaries of this region. Genomic sequences homologous to the 5-kb mRNA span over 75 kb of the amplified DNA segment. The level of expression of this mRNA in multidrug-resistant cells is correlated with the degree of gene amplification and the degree of drug resistance. Our results strongly suggest that the 5-kb mRNA species plays a role in the mechanism of multidrug resistance common to the LZ and C5 cell lines.

Structural analysis of the X-linked gene encoding human glucose 6-phosphate dehydrogenase.

Abstract: We report the isolation and analysis of human genomic DNA clones spanning about 100 kb of the X chromosome and comprising the entire gene coding for the enzyme glucose 6-phosphate dehydrogenase (G6PD). The G6PD gene is 18 kb long and consists of 13 exons: the protein-coding region is divided into 12 segments ranging in size from 12 to 236 bp; an intron is present in the 5' untranslated region. Mature G6PD mRNA has a single polyadenylation site in HeLa cells. The major 5' end of mature G6PD mRNA in several cell lines is located 177 bp upstream of the translation initiating codon; longer mRNA molecules extending further in the 5' direction could be identified by S1 mapping and by comparing genomic and cDNA sequences. The DNA sequence around the major mRNA start is very GC rich; as to putative transcription regulatory sequences, a non-canonical TATA box and 9 CCGCCC elements are present, but no CAAT element could be identified. The genomic DNA we have isolated includes another ubiquitously transcribed region, provisionally named the GdX gene. Although the function of GdX is as yet unknown, we have established that this gene is located about 40 kb downstream of G6PD and is transcribed in the same direction. A comparative analysis of the promoter region of G6PD and 10 other housekeeping enzyme genes has confirmed the presence of a number of common features. In particular, in the eight cases in which a 'TATA' box is present, a conserved sequence of 25 bp is seen immediately downstream.

Complete nucleotide sequence of the gene for human interleukin 1 alpha.

Abstract: The chromosomal gene for human interleukin 1 alpha (IL-1 alpha) was isolated from a human genomic DNA library by using as a probe cloned human IL-1 alpha cDNA. Complete nucleotide sequence of about 12 kilobase pairs (kbp) long was determined and the structure and organization of this gene were elucidated. This gene contains seven exons and six introns. The first exon encodes the 5'-untranslated region. Most of the prepeptide portion of the precursor polypeptide is encoded by the next three exons, and the mature form of IL-1 alpha is encoded by the remaining three exons. The last exon also encodes the intronless 3'-untranslated region. The fourth intron as well as both 5'- and 3'-flanking regions contains the sequence of an Alu family member. In the middle part of the last intron, a 46-bp sequence with a unique structure is repeated five times in a head to tail manner. These repeats are flanked by the regions containing alternative purine and pyrimidine tracts. In the 5'-flanking region, immediately upstream of the putative TATA box, the 16-bp sequence highly homologous to the binding site of the adenovirus 2 major late promoter transcription factor is identified. The nucleotide sequence reported here showed only one nucleotide substitution in the 3'-untranslated exon in comparison with the nucleotide sequence of the cDNA from HL-60, a promyelocytic leukemia cell line, previously cloned and sequenced in our laboratories.

Caenorhabditis elegans DNA does not contain 5-methylcytosine at any time during development or aging

Abstract: DNA, isolated from age-synchronous senescent populations of Caenorhabditis elegans has been quantitatively and qualitatively analyzed for the presence of 5-methylcytosine. High performance liquid chromatography on two wild-type and several mutant strains of C. elegans failed to detect any 5-methylcytosine. The restriction endonuclease isoschizomers, HpaII and MspI, were used to digest genomic DNA after CsCl purification and failed to detect any 5' cytosine methylation at any age. We conclude that C. elegans does not contain detectable (0.01 mole percent) levels of 5-methylcytosine.

Long-range restriction site mapping of mammalian genomic DNA.

Abstract: Molecular analysis of many problems in genetics would be facilitated by the ability to construct restriction site maps of long stretches of genomic DNA and to directly place genes on these maps. Pulsed-field gradient gel electrophoresis allows measurement of the size of DNA fragments up to at least 2,000 kilobase pairs (kb) long and we have used this technique here to map sites for one class of infrequently cutting restriction enzyme over a total of 1,500 kb of mouse genomic DNA. The sites for these enzymes tend to be clustered in the genome. These clusters may correspond to the short stretches of C + G-rich unmethylated DNA often associated with mammalian genes.

High-frequency transfection of CHO cells using polybrene.

Abstract: High-frequency transfection of CHO cells has been achieved for several plasmids, a cosmid library, and genomic DNA using Polybrene and dimethyl sulfoxide. All plasmid transfectants examined were stable and exhibited plasmid sequences in genomic DNA. The method is simple, reproducible, and succeeded with several independent CHO clones in the presence or the absence of carrier DNA, even at very low concentrations of plasmid DNA.

Primary structure of the gene encoding the bifunctional dihydrofolate reductase-thymidylate synthase of Leishmania major.

Abstract: We have determined the nucleotide sequence of the dihydrofolate reductase-thymidylate synthetase (DHFR-TS) gene of the protozoan parasite Leishmania major (dihydrofolate reductase, EC 1.5.1.3 and thymidylate synthase, EC 2.1.1.45). The DHFR-TS protein is encoded by a single 1560-base-pair open reading frame within genomic DNA, in contrast to vertebrate DHFRs or mouse and phage T4 TSs, which contain intervening sequences. Comparisons of the DHFR-TS sequence with DHFR and TS sequences of other organisms indicate that the order of enzymatic activities within the bifunctional polypeptide chain is DHFR followed by TS, the Leishmania bifunctional DHFR-TS evolved independently and not through a phage T4-related intermediate, and the rate of evolution of both the DHFR and TS domains has not detectably changed despite the acquisition of new functional properties by the bifunctional enzyme. The Leishmania gene is 86% G+C in the third codon position, in contrast to genes of the parasite Plasmodium falciparum, which exhibit an opposite bias toward A+T. The DHFR-TS locus is encoded within a region of DNA amplified in methotrexate-resistant lines, as previously proposed.

The Triosephosphate lsomerase Gene from Maize: lntrons Antedate the Plant-Animal Divergence

Abstract: Mark Marchionni and Walter Gilbert Department of Cellular and Developmental Biology Harvard University Biological Laboratories 16 Divinity Avenue Cambridge, Massachusetts 02138 Summary We have cloned and characterized a cDNA and genomic DNA for the triosephosphate isomerase ex- pressed in maize roots. The gene is interrupted by eight introns. If we compare this gene with that for the protein in chicken, which has six intmns, we see that five of the introns are at identical places, one has shifted by three codons, and two are totally new. This great matching leads us to conclude that the introns were in place before the plant-animal divergence, and that the parental gene had at least eight introns, two of which were lost in the line that leads to animals. Introduction Genes of higher eukaryotes are discontinuous: regions of noncoding DNA (introns) separate the coding sequence into discrete segments (exons). Gilbert (1978) proposed that introns are vestigial DNA sequence, remnants of a recombination process that accelerated molecular evolu- tion by assembling new linkage groups from the exons, creating novel gene products. That hypothesis of exon shuffling predicts that positions of introns should portray the evolutionary history of a gene; the exons themselves would encode distinct functional elements (Gilbert, 1978, 1979), stably folding peptides (Blake, 1978) or compact modules (Go, 1981, 1983). Evidence in support of these ideas derives from molecular studies on genes and pro- teins as diverse as immunoglobulins (reviewed by Tone- gawa, 1983, and Honjo, 1983) collagen (Yamada et al., 1980) serum albumin-a-fetoprotein (Sargent et al., 1981; Eiferman et al., 1981) ovomucoid (Stein et al., 1980), glo- bins (Go, 1981) intermediate filaments (Marchuk et al., 1984; Balcarek and Cowan, 1985), lysozyme (Jung et al., 1980) and crystallins (Moormann et al,, 1983). Moreover, recent studies (Siidhof et al., 1985a, 1985b) comparing the gene structure of low density lipoprotein receptor with those of epidermal growth factor and blood coagulation factors 9 and 10 provide a dramatic example of exon shuf- fling in recent evolutionary history. Although there are a few exceptions (Kaine et al., 1983; Chu et al., 1984; Nellen et al., 1981; Miller, 1984), the con- spicuous absence of introns in bacteria and yeast poses questions as to their age and origins. Were introns present as the first genes formed? Or have they more recently taken up residence in genomic DNA by invading continu- ous gene sequences? Doolittle (1978) suggested that the genomes of primitive cells contained introns but that rap- idly dividing organisms lost them by streamlining in re- sponse to the selection pressure of rapid reproduction. The highly conserved, ubiquitous glycolytic enzymes most likely evolved completely before the archaebacteria-pro- karyotic-eukaryotic division and thus represent extremely ancient genes. In vertebrates, introns punctuate nonran- domly the sequences encoding chicken pyruvate kinase (Lonberg and Gilbert, 1985), chicken glyceraldehyde phosphate dehydrogenase (Stone et al., 1985) chicken triosephosphate isomerase (Straus and Gilbert, 1985b), as well human phosphoglycerate kinase (Michelson et al., 1985), suggesting that the introns were not inserted into preexisting genes. To push our view of one these glycolytic enzyme genes back in time, we have character- ized a gene encoding triosephosphate isomerase (EC 5.3.1.1, TIM) in maize in order to compare its structure to the chicken gene. A remarkable conservation of intron po- sitions between these distant relatives indicates that the introns were in place before the plant-animal divergence, more than one billion years ago. Results Cloning and Structure of Maize TIM cDNA During glycolysis, triosephosphate isomerase (TIM) cata- lyzes the interconversion of dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. In addition to a cytosolic TIM, plant cells active in photosynthesis express a plastid enzyme encoded in the nucleus (Pichersky and Gottlieb, 1982). That isozyme functions in the dark reactions that fix carbon dioxide into sugars. As is true for other pho- tosynthetic proteins, the chloroplast-specific TIM is light inducible (e.g., Nelson et al., 1984; Berry-Lowe et al., 1982). Hence, mRNA from roots grown in the dark should be enriched in the cytosolic form, which ought to cog- nate to the chicken enzyme. Because TIM is less than 60% diverged across the most distant species (Pichersky et al., 1984; Straus and Gilbert, 1985b), cross-hybridiza- tion from chicken to maize root cDNA was a plausible ap- proach to cloning the maize gene. Using a chicken probe (Straus et al., 1985a), we screened 80,000 phage cDNA recombinants that repre- sented mRNA extracted from maize root seedlings grown in the dark for 7 days. We chose hybridization and wash- ing conditions of medium stringency (see Experimental Procedures) and detected 18 potential TIM clones. After partially mapping all of the inserts, we focused on two large (>800 bp) overlapping clones, subcloned each of these into the EcoRl site of pUC8 (Vieira and Messing, 1982), and sequenced (Maxam Gilbert, 1980) both strands of the DNA. Figure 1 displays a partial restriction map and the com- plete nucleotide sequence of a full-length maize root TIM cDNA clone, designated pMRT1. Within the 123 bases of 5’ untranslated sequence is a 37 base pyrimidine tract, which is flanked by two overlapping, imperfect, direct repeats of 20 bases each. A similar alternating pyrimidine

Primary structure of human ribosomal protein S14 and the gene that encodes it.

Abstract: Chinese hamster ribosomal protein S14 cDNA was used to recognize homologous human cDNA and genomic clones Human and Chinese hamster S14 protein sequences deduced from the cDNAs are identical Two overlapping human genomic S14 DNA clones were isolated from a Charon 28 placental DNA library A fragment of single-copy DNA derived from an intron region of one clone was mapped to the functional RPS14 locus on human chromosome 5q by using a panel of human X Chinese hamster hybrid cell DNAs The human S14 gene consists of five exons and four introns spanning 59 kilobase pairs of DNA Polyadenylated S14 transcripts purified from HeLa cell cytoplasma display heterogeneous 5' ends that map within noncoding RPS14 exon 1 This precludes assignment of a unique 5' boundary of RPS14 transcripts with respect to the cloned human genomic DNA Apparently HeLa cells either initiate transcription at multiple sites within RPS14 exon 1, or capped 5' oligonucleotides are removed from most S14 mRNAs posttranscription In contrast to the few murine ribosomal protein and several other mammalian housekeeping genes whose structures are known, human RPS14 contains a TATA sequence (TATACTT) upstream from exon 1 Three related short sequence motifs, also observed in murine and yeast ribosomal protein genes, occur in this region of the RPS14 gene RPS14 introns 3 and 4 both contain Alu sequences Interestingly, the Alu sequence in intron 3 is located slightly downstream from a chromosome 5 deletion breakpoint in one human X hamster hybrid clone analyzed

Molecular characterization of cell cycle gene CDC7 from Saccharomyces cerevisiae.

Abstract: The product of the CDC7 gene of Saccharomyces cerevisiae appears to have multiple roles in cellular physiology. It is required for the initiation of mitotic DNA synthesis. While it is not required for the initiation of meiotic DNA replication, it is necessary for genetic recombination during meiosis and for the formation of ascospores. It has also been implicated in an error-prone DNA repair pathway. Plasmids capable of complementing temperature-sensitive cdc7 mutations were isolated from libraries of yeast genomic DNA in the multicopy plasmid vectors YRp7 and YEp24. The complementing activity was localized within a 3.0-kilobase genomic DNA fragment. Genetic studies that included integration of the genomic insert at or near the CDC7 locus and marker rescue of four cdc7 alleles proved that the cloned fragment contains the yeast chromosomal CDC7 gene. The RNA transcript of CDC7 is about 1,700 nucleotides. Analysis of the nucleotide sequence of a 2.1-kilobase region of the cloned fragment revealed the presence of an open reading frame of 1,521 nucleotides that is presumed to encode the CDC7 protein. Depending on which of two possible ATG codons initiates translation, the calculated size of the CDC7 protein is 58.2 or 56 kilodaltons. Comparison of the predicted amino acid sequence of the CDC7 gene product with other known protein sequences suggests that CDC7 encodes a protein kinase.

Molecular cloning and complete primary sequence of human erythrocyte porphobilinogen deaminase.

Abstract: We have cloned and sequenced a cDNA clone coding for human erythrocyte porphobilinogen deaminase. It encompasses the translated region, part of the 5' and the 3' untranslated regions. The deduced 344 amino acid sequence is consistent with the molecular weight and the partial amino-acid sequence of the NH2 terminal of the purified erythrocyte enzyme. Southern analysis of human genomic DNA shows that its gene is present as a single copy in the human genome and Northern analysis demonstrates the presence of a single size species of mRNA in erythroid and non-erythroid tissues and in several cultured cell lines. Quantitative determinations indicate that the amount of PBG-D mRNA is modulated both by the erythroid nature of the tissue and by cell proliferation, probably at the transcriptional level.

Specific DNA probe for the diagnosis of Plasmodium falciparum malaria.

Abstract: Malaria can be diagnosed either by direct microscopic examination of blood smears, which is time consuming and requires expertise, or by immunological techniques, which are effective but do not distinguish between past and present infections. In this study, a simple procedure was developed for spotting lysed blood from infected patients directly onto nitrocellulose paper and identifying the malaria species on the basis of hybridization of parasite DNA with a species-specific probe. A genomic DNA library of Plasmodium falciparum was screened to detect clones containing DNA sequences that are highly repeated within the parasite genome. Several such clones were further analyzed to identify those that hybridize specifically with P. falciparum DNA but not with DNA from humans, P. vivax, or P. cynomolgi. This technique appears to be sensitive enough to detect 10 picograms of purified P. falciparum DNA (equivalent to 100 parasites) and in field studies is able to detect approximately 40 parasites per microliter of blood.

Transforming activity of human papillomavirus type 16 DNA sequence in a cervical cancer.

Abstract: A genomic DNA sample from cervical cancer tissue, containing human papillomavirus (HPV) type 16, was found to induce malignant transformation of NIH 3T3 cells when it was tested by transfection assays using the calcium phosphate coprecipitation technique. The primary and secondary transformants contained the HPV type 16 DNA sequences and human specific Alu family sequences. To the best of our knowledge, it has not been reported previously that HPV type 16 DNA sequences in total genomic DNA from a cervical cancer have transforming activity.

Putative diazepam binding inhibitor peptide: cDNA clones from rat.

Abstract: cDNA clones corresponding to the polypeptide that has been shown to be an endogenous diazepam binding inhibitor and may act as a physiological ligand for the benzodiazepine/beta-carboline receptor have been isolated from bacteriophage lambda recombinant libraries from rat hypothalamus, total brain, and liver. The clones contain an open reading frame corresponding to 87 amino acids. A signal sequence is not present. In addition to high levels of mRNA in various brain regions, RNA blot analysis reveals an abundance of diazepam binding inhibitor mRNA in many peripheral organs (e.g., testes, kidney, liver, and heart) that are known to be rich in peripheral benzodiazepine recognition sites. The size of the mRNA in all tissue examined is approximately 0.7 kilobase. Southern blot analysis of genomic DNA suggests the presence of about six genes in the rat, some of which may be pseudogenes.