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PubMed1991

Sequence analysis of DNA from formalin-fixed, paraffin-embedded human malignant melanoma.

Direct sequence analysis of in vitro amplified DNA from paraffin-embedded tissue is a promising new technique useful for detailed molecular analyses of material stored in dermatopathological archives. As only minute amounts of highly damaged DNA can be recovered from paraffin-embedded sections, sequence analysis of DNA from formalin-fixed and embedded tissue can result in less clear and readable data. We report here useful modifications that result in clear and readable sequence data. As an example, we detail the detection of a point mutation at codon 61 of the human N-ras gene in DNA from a formalin-fixed paraffin-embedded malignant melanoma specimen. This technique will be of value in many areas of molecular dermatopathology.

Base Sequence↗
PubMed2001

Identification of the nik gene cluster of Brucella suis: regulation and contribution to urease activity.

Analysis of a Brucella suis 1330 gene fused to a gfp reporter, and identified as being induced in J774 murine macrophage-like cells, allowed the isolation of a gene homologous to nikA, the first gene of the Escherichia coli operon encoding the specific transport system for nickel. DNA sequence analysis of the corresponding B. suis nik locus showed that it was highly similar to that of E. coli except for localization of the nikR regulatory gene, which lies upstream from the structural nikABCDE genes and in the opposite orientation. Protein sequence comparisons suggested that the deduced nikABCDE gene products belong to a periplasmic binding protein-dependent transport system. The nikA promoter-gfp fusion was activated in vitro by low oxygen tension and metal ion deficiency and was repressed by NiCl(2) excess. Insertional inactivation of nikA strongly reduced the activity of the nickel metalloenzyme urease, which was restored by addition of a nickel excess. Moreover, the nikA mutant of B. suis was functionally complemented with the E. coli nik gene cluster, leading to the recovery of urease activity. Reciprocally, an E. coli strain harboring a deleted nik operon recovered hydrogenase activity by heterologous complementation with the B. suis nik locus. Taking into account these results, we propose that the nik locus of B. suis encodes a nickel transport system. The results further suggest that nickel could enter B. suis via other transport systems. Intracellular growth rates of the B. suis wild-type and nikA mutant strains in human monocytes were similar, indicating that nikA was not essential for this step of infection. We discuss a possible role of nickel transport in maintaining enzymatic activities which could be crucial for survival of the bacteria under the environmental conditions encountered within the host.

ATP-Binding Cassette Transporters↗
PubMed1998

Isolation and characterization of Staphylococcus aureus starvation-induced, stationary-phase mutants defective in survival or recovery.

Ten Staphylococcus aureus mutants, defective in the starvation-induced stationary phase of growth were isolated from two independent Tn917-LTV1 transposon insertion libraries and were designated suv as they had apparent survival defects. Seven of these mutants were defective under amino-acid-limiting conditions alone. Two mutants (suv-3 and suv-20) demonstrated lower plating efficiency when starved for glucose, phosphate or amino acids and one mutant (suv-11) had reduced plating efficiency after amino acid or glucose starvation. All of the mutants tested were as resistant to hydrogen peroxide assault as the parent, but six were more sensitive to low pH conditions. All the mutants were physically mapped on the S. aureus chromosome using PFGE. Chromosomal DNA flanking the Tn917-LTV1 insertion sites was rescued by cloning into Escherichia coli. DNA sequence analysis resulted in the identification of a number of transposon-disrupted ORFs encoding putative components such as superoxide dismutase (suv-1), haem A synthase (suv-3), a component of the SOS response (suv-9) and hypoxanthine-guanine phosphoribosyltransferase (suv-20). The Tn917-LTV1 insertion created lacZ transcriptional fusions for some of the stationary-phase loci. Expression analysis indicated that suv-4 was induced at mid-exponential phase, whereas suv-3 and suv-11 were induced at the onset of stationary phase. The possible roles of these suv components in stationary-phase survival or recovery is discussed.

DNA Transposable Elements↗
PubMed1998

Molecular characterization of the human protein kinase C theta gene locus (PRKCQ).

Members of the protein kinase C (PKC) family of serine/threonine kinases, in particular PKCtheta, play critical roles in the regulation of differentiation and proliferation of T lymphocytes. In this study the genomic structure of the human PRKCQ gene that encodes PKCtheta was determined. Two genomic P1 clones were isolated from human P1 libraries using the PKCtheta cDNA as a probe and have been used to confirm the assignment of the single PRKCQ locus to chromosome 10p15 by FISH analysis. The PRKCQ locus, the first mammalian PKC gene locus characterized so far, spans approximately 62 kb and is composed of 15 coding exons and 14 introns, varying in size between 98 and 16000 bp. All exon-intron boundaries have been determined by long-range PCR and subsequent DNA sequence analysis. Comparison with other known genomic PKC genes reveals a high degree of homology to the genomic organization of the Drosophila melanogaster dPRKC gene. Alignment of the intron positions in the PRKCQ gene with the intron locations in the dPRKC gene indicates that the sites of seven of the 14 PRKCQ introns are exactly conserved. Exons 5 (32 bp), 11 (174 bp) and 12 (92 bp) share highest similarity in size, organization and primary structure with their counterparts in the Drosophila gene. On the basis of this knowledge of the genomic PRKCQ locus, a directed search for potential genetic polymorphisms and/or genetic abnormalities involved in human genetic disease(s) can now be initiated.

Animals↗
PubMed1996

Location, DNA sequence and transcriptional analysis of the DNA polymerase gene of orf virus.

Degenerate oligonucleotides representing conserved regions of various DNA polymerases hybridized to a region located 26 kb from the left end of the orf virus (OV) strain NZ-2 genome. DNA sequence analysis of this region revealed a 3024 bp open reading frame able to encode a protein with 56 percent amino acid identity to the DNA polymerase of vaccinia virus (VAC) and with significant homology to other DNA polymerases. Early transcripts derived from the open reading frame were detected in RNA purified from OV-infected cells, and 5' ends were mapped to a region 8-19 nt downstream from an A/T-rich sequence that resembles VAC early promoters. Unlike the VAC gene, the OV DNA polymerase makes almost exclusive use of G/C coding options. Attempts to substitute the activity of the OV DNA polymerase for its VAC counterpart were unsuccessful. This may indicate that the OV DNA polymerase is incompatible with VAC accessory proteins.

Amino Acid Sequence↗
PubMed2000

Malignant hypertrophic cardiomyopathy caused by the Arg723Gly mutation in beta-myosin heavy chain gene.

Mutations causing hypertrophic cardiomyopathy have been described in nine genes encoding sarcomeric proteins. We report a new mutation in three families, with a C-->G transversion in nucleotide 12 307 of the beta-myosin heavy chain gene, located at the essential light chain interacting region, resulting in the replacement of arginine by glycine at amino acid residue 723. PCR amplification of the selected regions followed by single strand conformation polymorphism analysis, DNA sequencing of the polymorphic patterns and restriction analysis were used to detect the mutation. A total of 23 individuals were diagnosed as carriers, and seven were obligate carriers or had been clinically diagnosed. The Arg723Gly mutation was associated with a malignant phenotype. Ten out of 30 affected members died suddenly or needed an implantable cardioverter-defibrillator at a mean age of 42, and seven members developed progressive heart failure, leading to death or heart transplant in five, at a mean age of 50 years. Echocardiography showed non-obstructive left ventricular hypertrophy in affected members older than 20 (sensitivity 68%). Mean survival of affected members was 51 years. In conclusion, a new mutation Arg723Gly in beta-myosin heavy chain gene is reported which shortens life expectancy because of sudden death and end-stage heart failure.

Adult↗
PubMed2000

Chromosome mapping of the human genes encoding the MAP kinase kinase MEK1 (MAP2K1) to 15q21 and MEK2 (MAP2K2) to 7q32.

Activation of the ERK mitogen-activated protein (MAP) kinase pathway has been implicated in the regulation of cell growth, differentiation and senescence. In this pathway, the MAP kinases ERK1/ERK2 are phosphorylated and activated by the dual-specificity kinases MEK1 and MEK2, which in turn are activated by serine phosphorylation by a number of MAP kinase kinase kinases. We report here the chromosomal localization of the human genes encoding the MAP kinase kinase isoforms MEK1 and MEK2. Using a combination of fluorescence in situ hybridization, somatic cell hybrid analysis, DNA sequencing and yeast artificial chromosome (YAC) clone analysis, we have mapped the MEK1 gene (MAP2K1) to chromosome 15q21. We also present evidence for the presence of a MEK1 pseudogene on chromosome 8p21. The MEK2 gene (MAP2K2) was mapped to chromosome 7q32 by fluorescence in situ hybridization and YAC clone analysis.

Blotting, Southern↗
PubMed2006

Comparison of body weight and gene expression in amelogenin null and wild-type mice.

Amelogenin (AmelX) null mice develop hypomineralized enamel lacking normal prism structure, but are healthy and fertile. Because these mice are smaller than wild-type mice prior to weaning, we undertook a detailed analysis of the weight of mice and analyzed AmelX expression in non-dental tissues. Wild-type mice had a greater average weight each day within the 3-wk period. Using reverse transcription-polymerase chain reaction (RT-PCR), products of approximately 200 bp in size were generated from wild-type teeth, brain, eye, and calvariae. DNA sequence analysis of RT-PCR products from calvariae indicated that the small amelogenin leucine-rich amelogenin peptide (LRAP), both with and without exon 4, was expressed. No products were obtained from any of the samples from the AmelX null mice. We also isolated mRNAs that included AmelX exons 8 and 9, and identified a duplication within the murine AmelX gene with 91% homology. Our results add additional support to the hypothesis that amelogenins are multifunctional proteins, with potential roles in non-ameloblasts and in non-mineralizing tissues during development. The smaller size of AmelX null mice could potentially be explained by the lack of LRAP expression in some of these tissues, leading to a delay in development.

Amelogenin↗
PubMed1993

Design of Onchocerca DNA probes based upon analysis of a repeated sequence family.

Repeated DNA sequences have been instrumental in the development of DNA probes for many different parasites. Isolation of such DNA probes has generally been accomplished by differential screening of genomic libraries with total genomic DNA preparations. In the current work, a rational design strategy is presented for the development of oligonucleotide probes based upon repeated sequence families. A repeated sequence family present in the genome of Onchocerca parasites, designated O-150, has been amplified from various samples of genomic DNA using PCR. DNA sequence analysis of the resulting PCR products demonstrated that the sequences may be arranged into clusters within which the individual sequences are identical or nearly identical. Differences among the cluster consensus sequences have been exploited to explain the specificities of previously isolated O-150 based probes and to develop two new oligonucleotide probes. One of these probes hybridizes specifically to Onchocerca volvulus O-150 PCR products, while the second hybridizes specifically to O-150 PCR products from the closely related bovine parasite O. ochengi. These oligonucleotide probes have been used to characterize Onchocerca infective larvae isolated from wild caught infected flies in West Africa. Because repeated sequence families are a common feature of most genomes, including those of parasites, this method should be applicable to the rational design of oligonucleotide probes for other parasitic infections.

Animals↗
PubMed1998

Inactivating mutations in the 25-hydroxyvitamin D3 1alpha-hydroxylase gene in patients with pseudovitamin D-deficiency rickets.

BACKGROUND: Pseudovitamin D-deficiency rickets is characterized by the early onset of rickets with hypocalcemia and is thought to be caused by a deficit in renal 25-hydroxyvitamin D3 1alpha-hydroxylase, the key enzyme for the synthesis of 1alpha,25-dihydroxyvitamin D3. METHODS: We cloned human 25-hydroxyvitamin D3 1alpha-hydroxylase complementary DNA (cDNA) using a mouse 1alpha-hydroxylase cDNA fragment as a probe. Its genomic structure was determined, and its chromosomal location was mapped by fluorescence in situ hybridization. We then identified mutations in the 1alpha-hydroxylase gene in four unrelated patients with pseudovitamin D-deficiency rickets by DNA-sequence analysis. Both the normal and the mutant 1alpha-hydroxylase proteins were expressed in COS-1 cells and were assayed for 1alpha-hydroxylase activity. RESULTS: The gene for 25-hydroxyvitamin D3 1alpha-hydroxylase was mapped to chromosome 12q13.3, which had previously been reported to be the locus for pseudovitamin D-deficiency rickets by linkage analysis. Four different homozygous missense mutations were detected in this gene in the four patients with pseudovitamin D-deficiency rickets. The unaffected parents and one sibling tested were heterozygous for the mutations. Functional analysis of the mutant 1alpha-hydroxylase protein revealed that all four mutations abolished 1alpha-hydroxylase activity. CONCLUSIONS: Inactivating mutations in the 25-hydroxyvitamin D3 1alpha-hydroxylase gene are a cause of pseudovitamin D-deficiency rickets.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗
PubMed1995

Genetic analysis of the modABCD (molybdate transport) operon of Escherichia coli.

DNA sequence analysis of the modABCD operon of Escherichia coli revealed the presence of four open reading frames. The first gene, modA, codes for a 257-amino-acid periplasmic binding protein enunciated by the presence of a signal peptide-like sequence. The second gene (modB) encodes a 229-amino-acid protein with a potential membrane location, while the 352-amino-acid ModC protein (modC product) contains a nucleotide-binding motif. On the basis of sequence similarities with proteins from other transport systems and molybdate transport proteins from other organisms, these three proteins are proposed to constitute the molybdate transport system. The fourth open reading frame (modD) encodes a 231-amino-acid protein of unknown function. Plasmids containing different mod genes were used to map several molybdate-suppressible chlorate-resistant mutants; interestingly, none of the 40 mutants tested had a mutation in the modD gene. About 35% of these chlorate-resistant mutants were not complemented by mod operon DNA. These mutants, designated mol, contained mutations at unknown chromosomal location(s) and produced formate hydrogenlyase activity only when cultured in molybdate-supplemented glucose-minimal medium, not in L broth. This group of mol mutants constitutes a new class of molybdate utilization mutants distinct from other known mutants in molybdate metabolism. These results show that molybdate, after transport into cells by the ModABC proteins, is metabolized (activated?) by the products of the mol gene(s).

Amino Acid Sequence↗
PubMed1996

A member of the phylogenetically conserved CAD family of transcriptional regulators is dramatically up-regulated during the programmed cell death of skeletal muscle in the tobacco hawkmoth Manduca sexta.

The intersegmental muscles (ISMs) of the tobacco hawkmoth Manduca sexta participate in the emergence behavior of the adult moth at the end of metamorphosis and then die during the subsequent 30-hr period. The trigger for this death is a decline in the circulating titer of the insect molting hormone 20-hydroxyecdysone (20-HE). Previous work has demonstrated that the ability of the ISMs to die is dependent on new gene expression. Using a differential hybridization cloning strategy, a cDNA library made from the ISMs committed to die was screened, and four up-regulated clones were isolated. One clone, 18-56, was selected for this study. Northern and Western analysis demonstrated that while clone 18-56 was expressed in all tissues examined and during every stage of ISM development, there was a dramatic increase in expression at both mRNA and protein levels when the ISMs became committed to die. If ISM death was delayed by an injection of 20-HE on the day proceeding adult emergence, 18-56 expression remained at basal levels. Immunocytochemistry demonstrated that 18-56 protein was located predominantly in nuclei prior to the commitment of the ISMs to die and then accumulated to high levels in cytoplasm at the time of cell death. DNA sequence analysis revealed that 18-56 protein shares 74% identity with yeast SUG1 and 92% with human Trip1, both of which are members of the conserved CAD (Conserved ATPase-containing Domain) family of putative transcriptional regulators. To verify that these genes shared functional as well as sequence homology, Manduca clone 18-56 was transformed into a yeast mutant for SUG1 function. Manduca 18-56 was able to both complement the lethal SUG1 phenotype and to suppress the transcriptional activity of a SUG1 mutation in yeast. Taken together, these data support the hypothesis that members of the phylogenetically conserved CAD family participate in important basal and developmental processes.

ATPases Associated with Diverse Cellular Activitie↗
PubMed2001

Serine acetyltransferase involved in cysteine biosynthesis from spinach: molecular cloning, characterization and expression analysis of cDNA encoding a plastidic isoform.

A cDNA clone that encodes a chloroplast-localizing isoform of serine acetyltransferase (SATase) (EC 2.3.1.30) was isolated from spinach (Spinacia oleracea L.). The cDNA encodes a polypeptide of 347 amino acids containing a putative transit peptide of ca. 60-70 amino acids at the N-terminal. Deduced amino acid sequence of SATase from spinach exhibited homology with other SATases from plants. DNA blot hybridization analysis showed the presence of 2-3 copies of Sat gene in the genome of spinach. RNA blot hybridization analysis indicated the constitutive expression of Sat gene in green and etiolated seedlings of spinach. Bacterial expression of the cDNA could directly rescue the cysteine auxotrophy of Escherchia coli caused by a lack of SATase locus (cysE). Catalytically active SATase protein was produced in E. coli cells. L-Cysteine, an end product of the cysteine biosynthetic pathway, inhibited the activity of recombinant spinach SATase, indicating the regulatory function of SATase in this metabolic pathway. A chloroplastic localization of this spinach SATase was revealed by the analyses of transgenic plant expressing transit peptide of SATase-beta-glucuronidase (GUS) fusion protein, and transient expression using the transit peptide-green fluorescent protein (GFP) fusion protein. The result from in vitro translation analysis suggests that this cDNA may encode both plastidic and cytosolic SATases.

Acetyltransferases↗
PubMed1987

Sequence and transcription analysis of the human cytomegalovirus DNA polymerase gene.

DNA sequence analysis has revealed that the gene coding for the human cytomegalovirus (HCMV) DNA polymerase is present within the long unique region of the virus genome. Identification is based on extensive amino acid homology between the predicted HCMV open reading frame HFLF2 and the DNA polymerase of herpes simplex virus type 1. We present here a 5280-base-pair DNA sequence containing the HCMV pol gene, along with the analysis of transcripts encoded within this region. Since HCMV pol also shows homology to the predicted Epstein-Barr virus pol, we were able to analyze the extent of homology between the DNA polymerases of three distantly related herpesviruses, HCMV, Epstein-Barr virus, and herpes simplex virus. The comparison shows that these DNA polymerases exhibit considerable amino acid homology and highlights a number of highly conserved regions; two such regions show homology to sequences within the adenovirus type 2 DNA polymerase. The HCMV pol gene is flanked by open reading frames with homology to those of other herpesviruses; upstream, there is a reading frame homologous to the glycoprotein B gene of herpes simplex virus type 1 and Epstein-Barr virus, and downstream there is a reading frame homologous to BFLF2 of Epstein-Barr virus.

Amino Acid Sequence↗
PubMed1995

Analysis of the 5' end of the rat plasma membrane Ca(2+)-ATPase isoform 3 gene and identification of extensive trinucleotide repeat sequences in the 5' untranslated region.

We have characterized the 5' end of the rat gene encoding isoform 3 of the plasma membrane Ca(2+)-ATPase using S1 nuclease protection and DNA sequence analysis. The 5'-untranslated region consists of over 900 nucleotides and includes a 217-nucleotide sequence composed of alternating tracts of TCC and ACC trinucleotides. Analysis of genomic sequences 5' to the transcription initiation site revealed potential binding sites for transcription factors that are active in muscle and brain.

Animals↗
PubMed1996

Molecular cloning and nucleotide sequence determination of three envelope genes of classical swine fever virus Taiwan isolate p97.

A strain of classical swine fever virus (CSFV) has been isolated in Taiwan. The cDNA coding for three envelope glycoproteins E1, E2 and E3 were molecularly cloned from purified viral particles using the reverse transcription-polymerase chain reaction (RT-PCR) method and sequence-specific primers. The resulting PCR products (1113 bp for E1. 699 bp for E2 and 567 bp for E3) were cloned into the SmaI site of pUC19 and then subjected to DNA sequence analysis. Data showed that nucleotide sequence of the three envelope genes shared a 82-83% homology with the corresponding genes of three other strains (Alfort, Brescia and Weybridge). However, the homology of the deduced amino acid sequence was greater than 90% among the four strains. The potential asparagine-linked glycosylation sites for E1 (5 sites), E2 (7 sites) and E3 (2 sites) were conserved. This suggests that the Taiwan p97 strain is distinct from other three strains described. The variations may have implications for future vaccine development. The sequence has been submitted to GenBank. The accession numbers are U43924 and U03290.

Amino Acid Sequence↗
PubMed1994

Detection of deletion mutations extending beyond the HPRT gene by multiplex PCR analysis.

A multiplex PCR assay was developed for the rapid analysis of deletion size at the hypoxanthine phosphoribosyltransferase (hprt) locus. The DNA sequence of mapped DNA segments flanking the hprt gene was determined. These cloned DNAs were derived from the ends of a set of overlapping yeast artificial chromosomes (YAC) defining a contig of 8 Mb at Xq26 and including hprt. We used "bubble" PCR to isolate an additional YAC end-clone. Seven primer pairs were derived from DNA sequence analysis of the clones and incorporated into a multiplex PCR assay. These primer pairs define loci located approximately 750 kb and 350 kb upstream of hprt and 300 kb, 540 kb, 900 kb, 1260 kb, and 1400 kb downstream of hprt. A primer pair for an unlinked and unselected gene sequence (K-ras) was also included in the multiplex reaction to serve as an internal positive control. Using this new assay, hprt mutant DNAs can be screened to determine the extent of deletion. Deletions larger than 2 Mb have been identified and show that large deletions can be tolerated at this hemizygous locus.

Adult↗
PubMed2005

Draft versus finished sequence data for DNA and protein diagnostic signature development.

Sequencing pathogen genomes is costly, demanding careful allocation of limited sequencing resources. We built a computational Sequencing Analysis Pipeline (SAP) to guide decisions regarding the amount of genomic sequencing necessary to develop high-quality diagnostic DNA and protein signatures. SAP uses simulations to estimate the number of target genomes and close phylogenetic relatives (near neighbors or NNs) to sequence. We use SAP to assess whether draft data are sufficient or finished sequencing is required using Marburg and variola virus sequences. Simulations indicate that intermediate to high-quality draft with error rates of 10(-3)-10(-5) (approximately 8x coverage) of target organisms is suitable for DNA signature prediction. Low-quality draft with error rates of approximately 1% (3x to 6x coverage) of target isolates is inadequate for DNA signature prediction, although low-quality draft of NNs is sufficient, as long as the target genomes are of high quality. For protein signature prediction, sequencing errors in target genomes substantially reduce the detection of amino acid sequence conservation, even if the draft is of high quality. In summary, high-quality draft of target and low-quality draft of NNs appears to be a cost-effective investment for DNA signature prediction, but may lead to underestimation of predicted protein signatures.

Computational Biology↗