Nucleotide Sequence Of Genomic DNA Research Articles

A Heterozygous Splice-Site Mutation in PTHLH Causes Autosomal Dominant Shortening of Metacarpals and Metatarsals.

Short metacarpals and/or metatarsals are typically observed in pseudohypoparathyroidism (PHP) type Ia (PHP1A) or pseudo-PHP (PPHP), disorders caused by inactivating GNAS mutations involving exons encoding the alpha-subunit of the stimulatory G protein (Gsα). Skeletal abnormalities similar to those in PHP1A/PPHP were present in several members of an extended Belgian family without evidence for abnormal calcium and phosphate regulation. Direct nucleotide sequencing of genomic DNA from an affected individual (190/III-1) excluded GNAS mutations. Instead, whole exome analysis revealed a novel heterozygous A>G change at nucleotide -3 upstream of PTHLH exon 3 that encodes the last two amino acids of the prosequence and the mature PTHrP. The same nucleotide change was also found in her affected mother and maternal aunt (190/II-2, 190/II-1), and her affected twin sons (190/IV-1, 190/IV-2), but not in her unaffected daughter (190/IV-3) and sister (190/III-2). Complementary DNA derived from immortalized lymphoblastoid cells from 190/IV-2 (affected) and 190/IV-3 (unaffected) was PCR-amplified using forward primers located either in PTHLH exon 1 (noncoding) or exon 2 (presequence and most of the prosequence), and reverse primers located in the 3'-noncoding regions of exons 3 or 4. Nucleotide sequence analysis of these amplicons revealed for the affected son 190/IV-2, but not for the unaffected daughter 190/IV-3, a heterozygous insertion of genomic nucleotides -2 and -1 causing a frameshift after residue 34 of the pre/prosequence and thus 29 novel residues without homology to PTHrP or any other protein. Our findings extend previous reports indicating that PTHrP haploinsufficiency causes skeletal abnormalities similar to those observed with heterozygous GNAS mutations. © 2018 American Society for Bone and Mineral Research.

Differential DNA damage response to UV and hydrogen peroxide depending of differentiation stage in a neuroblastoma model

DNA is a frequent target of oxidative damage, and DNA damage removal is therefore a crucial process in prevention of or recovery from degenerative diseases. DNA repair is an essential system for maintaining the inherited nucleotide sequence of genomic DNA over time. Cells engage in efficient DNA repair mechanisms, the activity of which can vary depending on the type of lesion and the developmental stage. Base excision repair (BER) and nucleotide excision repair (NER) are the major repair pathways addressed in this study. BER is the principal mechanism for repair of DNA oxidative lesions, while NER is the mechanism for repair of a variety of helix-distorting lesions such as those caused by UV radiation. Recent studies suggest that NER plays a cooperative role in removal of oxidative lesions. Little is known about the roles of DNA damage sensors and repair factors in terminally differentiated, non-proliferating cells such as neurons, which are vulnerable to oxidative damage from reactive oxygen species generated by endogenous or exogenous agents. We used the human neuroblastoma MSN cell model to investigate whether terminally differentiated neuronal cells respond to lesions cause in the DNA helix, such as UV-induced CPD and the major DNA oxidative lesion 8OHdG, and thereby clarify the role of NER capacity. We observed differences in DNA damage removal depending on the challenge insult and the differentiation state. Differentiated MSN cells, compared with undifferentiated cells, showed greater sensitivity to UVC and decreased DNA damage over time. In contrast, undifferentiated cells displayed genotoxicity induced by oxidative insult and tended to accumulate DNA damage and 8OHdG lesions over time. Our findings suggest the participation of GG-NER, TC-NER and BER proteins in the removal of 8-OHG and CPDs indicating a dynamic role in overall response to damage.

Exon 87 skipping of the COL7A1 gene in dominant dystrophic epidermolysis bullosa

Dystrophic epidermolysis bullosa (DEB) is a rare, inherited, blistering disorder resulting from mutations in the COL7A1 gene, which encodes the anchoring fibrils, type VII collagen. We herein describe a further Japanese girl diagnosed with dominant DEB (DDEB). She had blisters sporadically and erosions healed with mild scarring and milia on the knees and pretibial regions. Severe pruritus was present at this time. Direct nucleotide sequencing of genomic DNA disclosed a heterozygous same splice-site mutation c.6900G>A in the COL7A1, which causes in-frame exon 87 skipping. So far, five different COL7A1 mutations leading to exon 87 skipping have been identified in rare forms of DEB: four DDEB pruriginosa and one pretibial DDEB. Therefore, a recent study suggested that exon 87 skipping in COL7A1 was related to the phenotype of DDEB pruriginosa. When she was 18 years old, however, the blister formation and pruritus markedly decreased. Therefore, her clinical symptoms were consistent to very mild DDEB but not to DDEB pruriginosa. Taken together, in-frame exon 87 skipping through c.6900G>A mutation may account for the mild skin features, rather than DDEB pruriginosa, in the present case.

Penicillium marneffei SKN7, a Novel Gene, could Complement the Hypersensitivity of S. cerevisiae skn7 Disruptant Strain to Oxidative Stress

Penicillium marneffei is an intracellular fungal pathogen. Being resistant to the reactive oxygen species (ROS) produced by phagocytes is a key step to the surviving of P. marneffei within the phagocytes, as well as its invasion to the host. SKN7, a transcription factor, contributes to the oxidative stress response in yeasts. We cloned the SKN7 ortholog in P. marneffei and compared its transcription on both mycelial and yeast phase, identified its function by complement the S. cerevisiae skn7 disruptant strain. The result showed the full sequence of SKN7 gene was about 2.5 kb, open reading frame extended to 1,845 bp and encoded a putative protein of 614 amino acids. Comparative analysis of the nucleotide sequence of genomic DNA and cDNA confirmed the presence of four introns and two highly conserved HSF-DNA-bind and REC domain. The deduced amino acid sequence was homologous to SKN7 from other fungi. Further, P. marneffei cDNA can partly complement S. cerevisiae skn7 disruptant strain, which was not viable in the presence of 2.5 mM H2O2. The expression level of SKN7 on the two phases, however, had no difference. These results indicated that P. marneffei SKN7 could response to oxidative stress and it was not a phase-specific gene.

A highly efficient miPCR method for isolating FSTs from transgenic Arabidopsis thaliana plants

The exact localization of an insertion in the genome of transgenic plants obtained by Agrobacterium-mediated transformation is an integral part of most experiments aimed at studying these types of mutants. There are several methods for isolating unknown nucleotide sequences of genomic DNA which flank the borders of T-DNA integrated in the genome of plants. However, all the methods based on PCR have limitations which in some cases do not permit the desired objective to be achieved. We have developed a new technique for isolating flanking sequence tags (FSTs) via modified inverse PCR. This method is highly efficient and simple, but also retains the advantages of previously well-documented approaches.

Identification, cloning and sequencing of a novel stress inducible metallothionein gene from locally isolated Tetrahymena tropicalis lahorensis

A novel cadmium inducible metallothionein ( TMCd1) gene has been identified and sequenced from the locally isolated ciliate, Tetrahymena tropicalis lahorensis from industrial effluents . The TMCd1 gene encodes 471 nucleotides, with TGA as the stop codon and TAA coding for glutamine. This new gene is quite different from the previously reported MT genes in Tetrahymena pyriformis and Tetrahymena pigmentosa. However, it shows 78% homology with four different Cd–MT genes reported from Tetrahymena thermophila. A TATA box is located in the 5′ flanking region at nucleotide 34–38 upstream region of ATG. The TMCd1 gene is intronless like many other genes isolated from Tetrahymena species. The amino acids sequence of TMCd1 has a special feature of three CCCX 6CCX 6CCCX 6CC and two CCX 6CXCX 2CXCC intragenic tandem repeats with a conserved structural pattern of cysteine. The translated protein of TMCd1 contains 30.12% cysteine residues, which is a characteristic of a typical Tetrahymena Cd inducible MT genes. On the basis of 78% homology of nucleotide sequence of genomic DNA and its cDNA, TMCd1 has been considered as a new gene being reported from Tetrahymena tropicalis from this part of the world.

Prevalence of Long-QT Syndrome Gene Variants in Sudden Infant Death Syndrome

The hypothesis that some cases of sudden infant death syndrome (SIDS) could be caused by long-QT syndrome (LQTS) has been supported by molecular studies. However, there are inadequate data regarding the true prevalence of mutations in arrhythmia-susceptibility genes among SIDS cases. Given the importance and potential implications of these observations, we performed a study to more accurately quantify the contribution to SIDS of LQTS gene mutations and rare variants. Molecular screening of 7 genes (KCNQ1, KCNH2, SCN5A, KCNE1, KCNE2, KCNJ2, CAV3) associated with LQTS was performed with denaturing high-performance liquid chromatography and nucleotide sequencing of genomic DNA from 201 cases diagnosed as SIDS according to the Nordic Criteria, and from 182 infant and adult controls. All SIDS and control cases originated from the same regions in Norway. Genetic analysis was blinded to diagnosis. Mutations and rare variants were found in 26 of 201 cases (12.9%). On the basis of their functional effect, however, we considered 8 mutations and 7 rare variants found in 19 of 201 cases as likely contributors to sudden death (9.5%; 95% CI, 5.8 to 14.4%). We demonstrated that 9.5% of cases diagnosed as SIDS carry functionally significant genetic variants in LQTS genes. The present study demonstrates that sudden arrhythmic death is an important contributor to SIDS. As these variants likely modify ventricular repolarization and QT interval duration, our results support the debated concept that an ECG would probably identify most infants at risk for sudden death due to LQTS either in infancy or later on in life.

Mutation analysis in 51 patients with haemophilia A: report of 10 novel mutations and correlations between genotype and clinical phenotype

We report the results of genetic analysis on a series of 51 patients attending this Haemophilia Comprehensive Care Centre. The most common cause of severe haemophilia A--the factor VIII intron 22 inversion was detected in eight families and the factor VIII intron 1 inversion in three families. Mutation analysis was carried out on the remaining patients by nucleotide sequencing of genomic DNA after screening with conformation-sensitive gel electrophoresis (CSGE) or denaturing high-performance liquid chromatography (dHPLC). A total of 27 different FVIII non-inversion mutations were detected. Severe haemophilia was associated with 12 null mutations (six nonsense, six frameshift) and four missense mutations. A further 11 different missense mutations were associated with moderate or mild disease. To our knowledge, six null mutations [1950del 4(tttg), 3270-75insA, 4416del 10, 6735-38delA, W1029X, Y1792X] and four missense mutations (E1682K, M1947V, P2048L, P2143L) have not been previously published. Each novel missense mutation occurred at a highly conserved residue, no other candidate mutation was detected on screening the entire coding region of the FVIII gene and they were not detected in a screen of individuals without haemophilia A. The genotype-phenotype correlations of the FVIII mutations detected will be discussed.

Genomic structure and promoter activity of the testis haploid germ cell-specific intronless genes, Tact1 and Tact2.

The Tact1 and Tact2 genes, each of which encodes an actin-like protein, are exclusively expressed and translated in haploid germ cells in testis. To characterize the haploid germ cell-specific gene structure, a mouse genomic library was screened with a Tact1 cDNA as a probe, and four independent phage clones containing the Tact1 gene were isolated. Southern hybridization and sequencing analyses revealed that Tact1 and Tact2 were single copy genes contained on a common fragment in a head-to-head orientation, and that the distance between these genes was less than 2 kb. Comparison of the nucleotide sequences of genomic DNA and cDNA demonstrated that Tact1 and Tact2 lack introns, although all known actin or actin-related genes in mammals contain introns. Human Tact orthologues also lack introns and are located within 6.4 kb in a head-to-head orientation. These findings indicate that Tact1 and Tact2 or one of these genes arose by retroposition of a spliced mRNA transcribed from an actin progenitor gene prior to the divergence of rodents and primates. The Tact1 and Tact2 genes are unusual retroposons in that they have retained an open reading frame and are expressed in testicular germ cells, because almost all retroposons become pseudogenes. It was revealed that a 2kb sequence between the two genes bidirectionally controls haploid germ-cell specific expression by analyzing transgenic mice. Comparison of the murine Tact genes with their human orthologues showed a high level of identity between the two species in the 5'-upstream and non-coding sequences as well as in the coding region, indicating that conserved elements in these regions may be involved in the regulation of haploid germ cell-specific expression. The promoter region contains no TATA-, CCAAT- or GC-boxes, although there are potential cAMP response element (CRE)-like motifs in the 5'-upstream region and the 5'-untranslated region in Tact1 and Tact2, respectively. Transient promoter analyses indicate that CREMtau may activate Tact1 and Tact2 expression in germ cells.

Distribution and kainate-mediated induction of the DNA mismatch repair protein MSH2 in rat brain

DNA repair is one of the most essential systems for maintaining the inherited nucleotide sequence of genomic DNA over time. Repair of DNA damage would be particularly important in neurons, because these cells are among the longest-living cells in the body. MSH2 is one of the proteins which are involved in the recognition and repair of a specific type of DNA damage that is characterized by pair mismatches. We studied the distribution of MSH2 in rat brain by immunohistochemical analysis. We found the level of MSH2 expression in rat brain to be clearly heterogeneous. The highest intensity of staining was found in the pyramidal neurons of the hippocampus and in the entorhinal and frontoparietal cortices. Positive cells were observed in the substantia nigra pars compacta, in cerebellar granular and Purkinjie cells, and in the motor neurons of the spinal cord. We investigated the possible modulation of MSH2 expression after injection of kainate. Systemic administration of kainate induces various behavioural alterations and a typical pattern of neuropathology, with cell death in the hippocampal pyramidal neurons of the CA3/CA4 fields. Kainate injection also resulted in a marked, dose-dependent increase of MSH2 immunoreactivity in the hippocampal neurons of the CA3/CA4 fields. The effect was specific, since no changes in immunoreactivity were detected in the dentate gyrus nor in other brain areas. In summary, our data suggest that a mismatch DNA repair system, of which MSH2 protein is a representative component, is heterogeneously expressed in the rat brain and specifically induced by an experimental paradigm of excitotoxicity.

Cloning and Expression of Recombinant Human Growth/Differentiation Factor 5

The complete amino acid sequence of human Growth/differentiation factor 5 (huGdf5), a new member of the TGF-β superfamily, has been determined through initial degenerate PCR and subsequent cloning and nucleotide sequencing of genomic DNA and cDNA encoding the precursor and flanking regions. The huGdf5 gene consists of only two coding exons. The protein is highly homologous to its murine equivalent, particularly the mature part which differs only by one amino acid. Expression in HuTK− cells using recombinant vaccinia virus revealed the expected processed dimeric mature protein. Antibodies against huGdf5 were raised in chicken.

Identification of Intron/Exon Boundaries in Genomic DNA by Inverse PCR

This unit describes identifying intron/exon boundaries in genomic DNA by comparing nucleotide sequences of genomic DNA to cDNA. Cloned genomic DNA is prepared for inverse polymerase chain reaction (PCR) by digesting the DNA with a restriction enzyme and circularizing the restriction fragments by ligation. Diverging primer pairs for each exon are designed on the basis of the cDNA sequence. The circularized restriction fragments are amplified using these diverging primers, the PCR product is sequenced, and the sequence is compared to the cDNA sequence to determine the location of the intron/exon boundaries. The lower complexity of cloned DNA (e.g., YAC, P1, or cosmid DNA) facilitates preparation of good template. This unit describes identifying intron/exon boundaries in genomic DNA by comparing nucleotide sequences of genomic DNA to cDNA.

The Neurospora uvs-2 gene encodes a protein which has homology to yeast RAD18, with unique zinc finger motifs

A clone containing the DNA repair gene uvs-2 of Neurospora crassa was identified from a Neurospora genomic DNA library using the sib-selection method. Transformants were screened for resistance to methyl methane sulfonate (MMS). A DNA fragment that complements the uvs-2 mutation was subcloned by monitoring its ability to transform the uvs-2 mutant to MMS resistance. Deletion analysis of the cloned DNA indicated that the size of the uvs-2 gene is approximately 1.6 kb. The identity of the uvs-2 gene was verified by restriction fragment length polymorphism (RFLP) mapping. The sensitivity of the transformant to three different mutagens was similar to that of the wild-type strain. Nucleotide sequences of genomic DNA and cDNA of the uvs-2 gene indicated that it has an open reading frame (ORF) of 1572 bp with a 69 bp intron in the middle of the sequence. Two transcription initiation sites located around 73 bp and 290 bp upstream of the translation initiation codon were identified by primer extension experiments. Northern analysis revealed that the nature transcript of the uvs-2 gene was about 1.8 kb long. The uvs-2 gene ORF is deduced to encode a polypeptide of 501 amino acids with a molecular mass of 54 kDa. The proposed polypeptide has 25% identity to the RAD18 polypeptide of Saccharomyces cerevisiae and contains two unique zinc finger motifs for nucleic acid binding. Similarities between the phenotypes of the rad18 and uvs-2 mutants suggest that the uvs-2 gene encodes a protein which is involved in postreplication repair, rather than excision repair.

Structural characterization of exon 6 of the rat IGF-I gene.

In rat liver, insulin-like growth factor I (IGF-I) mRNAs exist as two major size classes of 7.5-7.0 kb and 1.2-0.9 kb. The 7.5- to 7.0-kb IGF-I mRNAs predominate in some nonhepatic tissues of the rat. Because the previously reported sequences of rat IGF-I cDNAs and genomic clones account for only 2.1 kb of sequence, the majority of the sequence of 7.5- to 7.0-kb rat IGF-I mRNAs was unknown. Using a combination of nucleotide sequencing of genomic DNA and cDNA clones and Northern hybridization and RNase protection, we have characterized a 6,354-base-long 3' exon (exon 6) of the rat IGF-I gene. The sequence of exon 6 establishes the previously unknown sequence of the 3' end of the 7.5- to 7.0-kb rat IGF-I mRNAs, comprised predominantly of an unusually long 3' untranslated sequence (3'UT). The long 3'UT contains multiple ATTTA, A(T)nA, and (T)nA sequences, as well as inverted repeats. These sequences may contribute to the shorter half-life of the 7.5- to 7.0-kb rat IGF-I mRNAs relative to the 1.2- to 0.9-kb forms that have been demonstrated previously in vitro and in vivo. We also demonstrate that the 7.5- to 7.0-kb rat IGF-I mRNAs are localized to the cytoplasm of rat liver, providing indirect evidence that they are mature and functional mRNAs.

Molecular cloning of a putative receptor protein kinase gene encoded at the self-incompatibility locus of Brassica oleracea.

Self-recognition between pollen and stigma during pollination in Brassica oleracea is genetically controlled by the multiallelic self-incompatibility locus (S). We describe the S receptor kinase (SRK) gene, a previously uncharacterized gene that resides at the S locus. The nucleotide sequences of genomic DNA and of cDNAs corresponding to SRK predict a putative transmembrane receptor having serine/threonine-specific protein kinase activity. Its extracellular domain exhibits striking homology to the secreted product of the S-locus glycoprotein (SLG) gene and is connected via a single pass transmembrane domain to a protein kinase catalytic center. SRK alleles derived from different S-locus genotypes are highly polymorphic and have apparently evolved in unison with genetically linked alleles of SLG. SRK directs the synthesis of several alternative transcripts, which potentially encode different protein products, and these transcripts were detected exclusively in reproductive organs. The identification of SRK may provide new perspectives into the signal transduction mechanism underlying pollen recognition.

Scientific and Standardization Committee Communications Recommendations for the Nomenclature of Mutant Fibrinolytic Genes and Their Proteins

1. Some of the guidelines on the nomenclature for the genotype of bacterial strains can be applied for recommendations on the nomenclature for the description of recombinant mutant fibrinolytic constructs. 2. In view of the usually different size of genomic DNA and corresponding cDNA, it is recommended to employ a distinct numbering system for nucleotide sequences of genomic DNA and of cDNA. Both numbering systems initiate at the utmost 5' terminus of mRNA synthesis; this 5' initiator nucleotide is assigned 1. 3. Amino-acid sequences are numbered with respect to the amino-terminus of the mature protein (amino-terminus is assigned 1). Amino-acid sequences constituting pre- and pro-sequences are numbered -1, -2, -3 etc. 4. Irrespective of the nature of the mutations, point mutations, deletions, insertions and substitutions within genomic DNA, cDNA and within a protein are described according to their sequential occurrence, according to the 5' to 3' direction for the nucleic acid and from the amino- to the carboxy-terminus for the protein, respectively. 5. It is recommended to describe the "genotype" of mutant genomic DNA or mutant cDNA, located on an appropriate vector, according to the following proposals: a) Point mutant: PSV2/t-PA D371A (Nomenclature for the vector pSV2 and t-PA has already been used throughout the literature. The designation of the point mutation is separated with a single "space" from the indication t-PA. The amino-acid substitution, due to a point mutation, is written in the one-letter code (italized sample). b) Deletion mutant:pSV2/t-PA del(S51-D87). (The abbreviation "del" indicates that a deletion has been constructed.(ABSTRACT TRUNCATED AT 250 WORDS)

Nucleotide sequence of a cloned hepatitis B virus genome, subtype ayr: comparison with genomes of the other three subtypes.

The entire nucleotide sequence of genomic DNA was determined for hepatitis B virus (HBV) of subtype ayr, which had been derived from the blood of a Japanese asymptomatic carrier. The genome was 3215 nucleotides long, and differed in DNA sequence by 10% from that of subtypes adw or ayw, but by only 2% from that of subtype adr. Amino acid sequences coded for by the S, C, P and X genes, as well as by the pre-S region, closely resembled those of subtype adr, indicating that the evolution of HBV/ayr from HBV/adr was more recent than the differentiation of the other three subtypes. In the product of the S gene, the mutually exclusive subtypic determinants of the surface antigen, d and y, were associated with variation of amino acid residues at only the 68th and 122nd positions from the N terminus, in contrast to the variation at as many as seven positions for the other set of subtypic determinants, w and r. Sequences representing high local hydrophilicity in the product of the S gene were involved in subtypic variation, although such sequences in the pre-S region were shared by HBV genomes of the various subtypes. In particular, a hydrophilic sequence of 19 amino acid residues, coded for by the pre-S(2) region and implicated in the presumed hepatotropism of HBV, was possessed in common by HBV/adr, HBV/ayr and HBV/ayw, and differed in HBV/adw by only one residue at the 9th position. This amino acid sequence appears to be a promising candidate for a synthetic peptide vaccine.

On the molecular mechanism of gypsy-induced mutations at the yellow locus of Drosophila melanogaster.

We determined the nucleotide sequence of genomic DNA corresponding to the yellow gene. The limits of the transcribed region were deduced from sequence analysis of yellow larval and pupal cDNA clones. The yellow transcription unit is simple, composed of two exons which are processed identically in both developmental stages into a mRNA of 1990 bp. The predicted yellow protein has a mol. wt of 60,752 daltons and appears to be a secreted protein having a structural function and not an enzymatic role in pigmentation. We also characterized the spontaneous mutation y2 and a revertant of this allele to investigate the mutagenic effect of the gypsy element inserted into this locus. Our results show that this transposon is inserted at -700 bp and that the y2+ revertant resulted from excision of the gypsy element leaving behind a complete long terminal repeat (LTR). We conclude, therefore, that the gypsy element is neither inserted into a pupal specific intron or regulatory sequence supporting the hypothesis that mutagenesis is a result of transcriptional interference by the gypsy element on the yellow gene.

Escherichia coli mutants defective in the γ subunit of proton-translocating ATPase: Intracistronic mapping of the defective site and the biochemical properties of the mutants

Various hybrid plasmids carrying a portion of the gene for the γ subunit of the H +-ATPase of Escherichia coli complemented five mutants defective in the enzyme in a genetic test, indicating that the mutants are defective in the γ subunit. Since the nucleotide sequence of genomic DNA carried on the plasmids is known, the defective site(s) of the mutants could be located within the gene for the γ subunit as follows: KF10 and NR70, KF1, and KF12 and KF13 have a mutation causing a defect(s) in amino acid residues 1 to 82, 83 to 167, and 168 to 287, respectively, of the γ subunit. The biochemical properties of all these mutants except NR70 were analyzed in terms of proton permeability of the membranes and assembly of F 1. Results suggested that KF1 and KF10 have defective F 1 without at least the α and β subunits on their membranes, whereas KF12 and KF13 have F 1's of rather similar structure to that of the wild type. Attempts were made to purify F 1 of KF12 as a single complex. Although the F 1 complex dissociated during purification, active α and β subunits of KF12 were partially purified. On the basis of these biochemical and genetic results, it is suggested that structural alterations in the primary sequence of the γ subunit corresponding to residues 1 to 167 cause more extensive defects in the assembly of F 1 than alteration in the sequence of residues 168 to 287.