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PubMed2002

Identification of truncated form of mouse HAX-1s gene (HAX-1xs) and characterization of its expression in small intestine and thymus of mice after burn injury.

Burn injury often leads to distant organ injury such as acute respiratory distress syndrome. We hypothesize that the pathophysiologic changes in distant organs result from orchestrated regulation of multiple genes in response to bum injury. Differential display was performed to identify genes regulated in distant organs in response to burn injury. Initial characterization of differentially amplified products demonstrated that HAX-1s mRNA was regulated in several distant organs after 18% total body surface area (TBSA) full-thickness flame burn injury in mice. Further characterization of HAX-1s mRNA revealed a novel transcript variant, which is rapidly and transiently induced in multiple tissues of mice within 6 h after burn injury. This novel HAX-1s transcript variant, called HAX-1xs, has an internal deletion of 252 nucleotides and single point mutation, resulting in reading frame intact. Western blot and immunohistochemical analyses of multiple tissues of mice using rabbit antibody raised against a 15-mer synthetic peptide clearly revealed the presence of HAX-1xs protein in the duodenum, and suggested that expression of HAX-1xs and/or HAX-1s was tissue- and cell type-specific. The expression of HAX-1xs and/or HAX-1s was distinctively regulated in Paneth cells of the duodenum and macrophages of the thymus after burn injury. These findings suggest that HAX-1xs has a different biological activity from HAX-1s and participates in a cascade of immediate-early cellular events in response to burn injury.

Adaptor Proteins, Signal Transducing↗

PubMed1983

Activation of the genes for variant surface glycoproteins 117 and 118 in Trypanosoma brucei.

We have studied the activation of genes for VSGs (variant surface glycoproteins) in Trypanosoma brucei (strain 427) in six independently isolated trypanosome clones; four expressing the gene for VSG 118 and two the gene for VSG 117. In all cases, gene activation is brought about by a duplicative transposition of the gene to an expression site located close to the end of a chromosome. The DNA segments flanking the expression-linked extra gene copy are nearly devoid of restriction enzyme recognition sites and their lengths vary by more than 10,000 base-pairs among different variants. From the correspondence of five upstream restriction sites, we conclude that the same expression site is used in each case. The transposition event does not lead to detectable alterations in the sequence coding for the mature protein. All restriction enzyme recognition sites detected in the basic copy gene are present also in each of the expression-linked copies. This argues against the introduction of mutations by an error-prone polymerase during the synthesis of the expression-linked copy. In five of the six variants, the 3' end of the VSG messenger RNA differs from that of the corresponding basic copy gene by multiple point mutations, insertions and deletions, starting at positions varying from 16 nucleotides upstream to 113 downstream of the last codon of the mature protein. We attribute this end alteration to the recombination process that introduces the gene into the expression site. We confirm that the expression-linked gene copy is more sensitive to DNase I than the corresponding basic copy gene. This appears to be due to its activated state and not to its location near the end of a chromosome, because another basic copy VSG gene permanently located near a chromosome end is not hypersensitive to DNase I. The mature transcripts of the 117 and 118 genes all possess the same 35 nucleotides at their 5' ends and these are not encoded contiguously in the basic gene copies with the remainder of the mRNAs. This extends our previous conclusion, that mature VSG mRNAs are formed by a splicing process in which the 35-nucleotide sequence encoded in the expression site is fused onto the body of the mRNA contributed by the transposed gene.

Animals↗

PubMed1994

Phenotypic and genotypic characterization of acyclovir-resistant varicella-zoster viruses isolated from persons with AIDS.

Phenotypic and genotypic analyses were done on 17 varicella-zoster virus (VZV) isolates recovered from 10 persons with AIDS (mean CD4 cell count, 16.4/mm3) who had chronic VZV lesions. Eleven acyclovir-resistant isolates were recovered from 10 patients after a mean of 20.1 weeks of therapy. Six susceptible isolates were recovered before acyclovir treatment (n = 1), early during therapy (n = 4; mean time, 4.2 weeks), or after discontinuation of acyclovir (n = 1). Acyclovir-resistant VZV isolates were deficient in thymidine kinase (TK) or induced a TK with altered substrate specificity; all isolates were susceptible to foscarnet. Ten of 11 acyclovir-resistant mutants contained tk gene mutations, including single nucleotide substitutions in highly conserved binding sites (n = 2) as well as nucleotide deletions (n = 4) and insertions (n = 4). These findings suggest that multiple, nonuniform mutations within the tk gene are associated with acyclovir-resistant VZV phenotypes.

AIDS-Related Opportunistic Infections↗

PubMed2000

A region of the transmembrane regulatory protein ToxR that tethers the transcriptional activation domain to the cytoplasmic membrane displays wide divergence among Vibrio species.

The virulence regulatory protein ToxR of Vibrio cholerae is unique in that it contains a cytoplasmic DNA-binding-transcriptional activation domain, a transmembrane domain, and a periplasmic domain. Although ToxR and other transmembrane transcriptional activators have been discovered in other bacteria, little is known about their mechanism of activation. Utilizing degenerate oligonucleotides and PCR, we have amplified internal toxR gene sequences from seven Vibrio and Photobacterium species and subspecies, demonstrating that toxR is an ancestral gene of the family Vibrionaceae. Sequence alignment of all available ToxR amino acid sequences revealed a region between the transcriptional activation and transmembrane domains that displays wide divergence among Vibrio species. We hypothesize that this region merely tethers the transcriptional activation domain to the cytoplasmic membrane and thus can tolerate wide divergence and multiple insertions and deletions. The divergence in the tether region at the nucleotide level may provide a useful tool for the distinction of Vibrio and Photobacterium species.

Amino Acid Sequence↗

PubMed2003

Progressive external ophthalmoplegia characterized by multiple deletions of mitochondrial DNA: unraveling the pathogenesis of human mitochondrial DNA instability and the initiation of a genetic classification.

Over the last decade, many sporadic and familial cases have been reported with multiple deletions of mitochondrial DNA (mtDNA) in postmitotic tissues. Most patients suffer from progressive external ophthalmoplegia (PEO) and may have a nuclear gene defect that predisposes to the accumulation of mtDNA deletions. Recently, positional cloning has led to the discovery of mutations in four such nuclear genes. Some mutations are dominant and others recessive. In all autosomal mutations, defective mtDNA replication and/or repair are probably responsible for the generation of secondary mtDNA deletions. There are also data suggestive of a prominent pathogenic role for disturbed nucleotide metabolism. We here present a tentative genotype-phenotype correlation. Since clinical presentations are heterogeneous and overlap with different previously described clinical syndromes, we advocate the use of a genetic, instead of a clinical, classification of disorders with multiple mtDNA deletions.

Animals↗

PubMed2006

The chimeric Arabidopsis CYCLIC NUCLEOTIDE-GATED ION CHANNEL11/12 activates multiple pathogen resistance responses.

To investigate the resistance signaling pathways activated by pathogen infection, we previously identified the Arabidopsis thaliana mutant constitutive expresser of PR genes22 (cpr22), which displays constitutive activation of multiple defense responses. Here, we identify the cpr22 mutation as a 3-kb deletion that fuses two cyclic nucleotide-gated ion channel (ATCNGC)-encoding genes, ATCNGC11 and ATCNGC12, to generate a novel chimeric gene, ATCNGC11/12. Genetic, molecular, and complementation analyses suggest that ATCNGC11/12, as well as ATCNGC11 and ATCNGC12, form functional cAMP-activated ATCNGCs and that the phenotype conferred by cpr22 is attributable to the expression of ATCNGC11/12. However, because overexpression of ATCNGC12, but not ATCNGC11, suppressed the phenotype conferred by cpr22, the development of this phenotype appears to be regulated by the ratio between ATCNGC11/12 and ATCNGC12. Analysis of knockout lines revealed that both ATCNGC11 and ATCNGC12 are positive mediators of resistance against an avirulent biotype of Hyaloperonospora parasitica. Through epistatic analyses, cpr22-mediated enhanced resistance to pathogens was found to require NDR1-dependent and EDS1/PAD4-dependent pathways. In striking contrast, none of these pathways was required for cpr22-induced salicylic acid accumulation or PR-1 gene expression. These results demonstrate that NDR1, EDS1, and PAD4 mediate other resistance signaling function(s) in addition to salicylic acid and pathogenesis-related protein accumulation. Moreover, the requirement for both NDR1-dependent and EDS1/PAD4-dependent pathways for cpr22-mediated resistance suggests that these pathways are cross-regulated.

Amino Acid Sequence↗

PubMed2001

Mitochondrial DNA polymorphisms in Thailand.

Nucleotide sequences of the D-loop region of human mitochondrial DNA from six small ethnic groups of Thailand i.e., Hill tribes (Lisu and Mussur), Phuthai, Lao Song, Chong, and aboriginal Sakai, were analyzed. The sequences were compared with those of native Thai populations from two provinces, Chiang Mai and Khon Kaen. Based on a comparison of the 563-bp sequences in 215 Thai individuals, 137 different sequence types were observed. Of these, 124 were unique to their respective populations, whereas 13 were shared between two to five populations. The intergenic COII/tRNALys 9-bp deletion was observed in every Thai population examined, except for the Sakai, with varying frequencies ranging from 18% to 40%. The D-loop sequences variation, and phylogenetic analysis, suggested that the 9-bp deletion had occurred in a very ancient ancestry of Southeast Asians, although multiple origins of the deletion cannot be ruled out. Genetic distances, based on net nucleotide diversities, between populations revealed that the Sakai were distantly related to the other Thai populations, while the Lao Song and Chong were closely related to each other. Close genetic affinities were also observed among the Hill tribes, Phuthai, and native northeast Thai (Khon Kaen), indicating that they may share some degree of the common ancestral maternal lineages.

Base Sequence↗

PubMed1982

Sequence analysis of cDNA's derived from the RNA of Sindbis virions and of defective interfering particles.

Sindbis virus generates defective interfering (DI) particles during serial high-multiplicity passage in cultured cells. These DI particles inhibit the replication of infectious virus and can be an important factor in the establishment and maintenance of persistent infection in BHK cells. In an effort to understand how these DI particles are generated and how they interfere with the replication of standard virus, we performed a partial sequence analysis of the RNA obtained from two independently isolated populations of DI particles and from two Sindbis virus variants and compared these with the RNA of the parental wild-type virus. The 3'-terminal regions of the RNAs were sequenced by the dideoxy chain terminating method. Internal regions of the RNA were examined by restriction endonuclease digestion of cDNA's made to the various RNAs and by direct chemical sequencing of 5' end-labeled restriction fragments from cDNA made to the DI RNAs. One of the variant viruses examined was originally derived from cells persistently infected with Sindbis virus for 16 months and is resistant to interference by the DI strains used. In the 3'-terminal region of the RNA from this variant, only two base changes were found; one of these occurs in the 20-nucleotide 3'-terminal sequence which is highly conserved among alphaviruses. The DI RNA sequences were found to have been produced not by a single deletional event, but by multiple deletion steps combined with sequence rearrangements; all sequences examined are derived from the plus strand of Sindbis virion RNA. Both DI RNAs had at least 50 nucleotides of wild-type sequence conserved at the 3' terminus; in addition, they both contained conserved and perhaps amplified sequences derived from the non-26S region of the genome which may be of importance in their replication and interference ability.

Base Sequence↗

PubMed2004

PXR (NR1I2): splice variants in human tissues, including brain, and identification of neurosteroids and nicotine as PXR activators.

To gain insight on the expression of pregnane X receptor (PXR), we analyzed PXR.1 and PXR alternatively spliced transcripts in a panel of 36 human tissues. PXR.1 was expressed in many more tissues than previously determined, including human bone marrow and select regions of the human brain. In each of these tissues, we observed alternative splicing of various exons of PXR that generated multiple distinct PXR isoforms. The most abundant PXR alternative mRNA transcripts lacked 111 nucleotides, deleting 37 amino acids from the PXR LBD (PXR.2), or lacked 123 nt, deleting 41 amino acids from the PXR LBD (PXR.3). CYP3A4, a gene transcriptionally regulated by PXR, showed incomplete overlap with PXR in its tissue distribution. Quantitation of PXR mRNAs in human liver demonstrated that PXR.2 and PXR.3 represented 6.7% and 0.32% of total PXR mRNA transcripts. Brain expression of PXR prompted analysis of whether some brain acting chemicals were PXR ligands. The neurosteroids allopregnanolone and pregnanolone activated PXR and induced transcription of a CYP3A4-luciferase reporter. Nicotine, the psychoactive and addictive chemical in cigarettes, and a known inducer of brain CYP2B6, was an efficacious activator of PXR and inducer of CYP3A4 transcription. Because nicotine activation of PXR will enhance metabolism of nicotine to the non-psychoactive cotinine, these results provide one molecular mechanism for the development of tolerance to nicotine. Moreover, the identification of PXR in many human tissues, such as brain, and activation by tissue specific ligands (such as neurosteroids) suggests additional biological roles for this receptor in these tissues.

Adult↗

PubMed2004

Deoxyribonucleotides and disorders of mitochondrial DNA integrity.

Mitochondrial DNA (mtDNA) depends on numerous nuclear encoded factors and a constant supply of deoxyribonucleoside triphosphates (dNTP), for its maintenance and replication. The function of proteins involved in nucleotide metabolism is perturbed in a heterogeneous group of disorders associated with depletion, multiple deletions, and mutations of the mitochondrial genome. Disturbed homeostasis of the mitochondrial dNTP pools are likely the underlying cause. Understanding of the biochemical and molecular basis of these disorders will promote the development of new therapeutic approaches. This article reviews the current knowledge of deoxyribonucleotide metabolism in relation to disorders affecting mtDNA integrity.

Animals↗

PubMed2006

IGHV gene insertions and deletions in chronic lymphocytic leukemia: "CLL-biased" deletions in a subset of cases with stereotyped receptors.

Nucleotide insertions/duplications or deletions in immunoglobulin heavy chain genes have been found in 24/760 patients (3.15%) with chronic lymphocytic leukemia (CLL). In 21/24 cases, the inserted/duplicated or lost nucleotides occurred in multiples of 3; therefore, the original reading frame was maintained and a potentially intact receptor was coded. The pattern and location of insertions/duplications or deletions in CLL and their restriction to mutated IGHV rearranged genes strongly suggests that they resulted from somatic hypermutation. Their incidence in CLL is consistent with previous reports in normal, auto-reactive and neoplastic human B cells, thus seemingly indicating that these modifications generally arise without any particular disease-specific associations. A striking exception to this rule was identified in CLL IGHV3-21-expressing cases: one amino acid was deleted from the CDR2 region in 16/63 (25.4%) mutated CLL IGHV3-21 sequences (including public database-derived IGHV3-21 CLL cases + the present series) vs. only 2/257 (0.78%) public database-derived mutated non-CLL IGHV3-21 sequences; 15/16 CLL IGHV3-21 sequences carrying this deletion belonged to a subset with unique, shared HCDR3 and light chain CDR3 motifs. This finding further supports the idea of selective antigenic pressures playing a pathogenetic role in some CLL cases.

Amino Acid Sequence↗

PubMed1986

Precise assignment of the light-strand promoter of mouse mitochondrial DNA: a functional promoter consists of multiple upstream domains.

Using deletion mutagenesis we localized the promoter for the light strand of mouse mitochondrial DNA to a 97-base-pair region, from -88 to +9 nucleotides of the transcriptional initiation site. Within this region the light-strand promoter could be dissected into at least three different functional domains. The specificity region, a maximum of 19 base pairs between -10 and +9 of the transcriptional initiation site, was essential and sufficient for accurate transcriptional initiation. A second region, extending to -29 nucleotides from the initiation site, facilitated the formation of a preinitiation complex between the template DNA and factor(s) present in the mitochondrial RNA polymerase fraction and was required for efficient transcription. A third, ill-defined upstream region, which extended up to -88 nucleotides from the initiation site, appeared to influence template transcriptional efficiencies in competition assays. Without the specificity domain, the upstream regions were incapable of supporting any transcription. The presence of multiple upstream domains was confirmed by disrupting nucleotide sequences in the upstream region by using linker insertion and linker replacement techniques.

Animals↗

PubMed1972

Sequence heterogeneity in closed simian virus 40 deoxyribonucleic acid.

The heteroduplex molecules formed by self-annealing of denatured, singly nicked simian virus 40 (SV40) deoxyribonucleic acid (DNA) prepared from closed viral DNA were examined by formamide-protein film electron microscopy to test the DNA for sequence homogeneity. Sequence inhomogeneity appears in the heteroduplexes as single-strand loops. These result from sequence deletion or from sequence substitution, if regions greater than 50 nucleotides are involved. The undenatured DNA from viruses passaged twice at multiplicities of infection much less than 1 plaque-forming unit (PFU) per cell appeared to be homogeneous in size. The heteroduplexes formed by this DNA indicated that approximately 2% of the molecules carried deletions, but that substitutions were below the level of detection. In contrast, undenatured DNA from viruses grown by passaging undiluted lysates seven times or by infection with stock virus at a multiplicity of infection of 5 PFU per cell contained a large frequency of molecules shorter than the full length. The heteroduplex samples indicated that 12 and 7% of the undenatured material contained base substitutions, and 13 and 11% contained deletions. The deletions and substitutions appear to occur in separate molecules. Length measurements on heteroduplexes displaying the loop characteristic of substitutions have established that these molecules are from true sequence substitutions, and not from adjacent or overlapping deletions. More than 80% of the molecules carrying substitutions are shorter than the native SV40 length. On the average, the substituted sequence is about 20% of the length of SV40, but it replaces a sequence about 30% of the native length. The substituted sequences may be host cell nuclear DNA, possibly arising from integration of SV40 into the chromosome followed by excision of the SV40 DNA together with chromosomal DNA.

Animals↗

PubMed1998

APC mutations in human colorectal adenomas: analysis of the mutation cluster region with temperature gradient gel electrophoresis and clinicopathological features.

To date, the earliest mutations in colorectal adenomas have been found in the adenomatous polyposis coli (APC) gene, considered the 'gatekeeper' in tumourigenesis. To study the types of APC gene mutations and their relation to clinicopathological features which are associated with malignant potential, the mutation cluster region of the APC gene was analysed in a series of 32 human sporadic colorectal adenomas from 22 patients. DNA was extracted from frozen sections and two overlapping fragments which cover the mutation cluster region were amplified using the polymerase chain reaction (PCR). Mutations were screened with temperature gradient gel electrophoresis and identified by DNA sequencing. Mutations were found in 14 samples (44 per cent) from 11 patients. The changes could be characterized as point mutations (n = 7), deletions of one or more nucleotides (n = 6), and insertions (n = 1). From five patients, multiple adenoma samples were obtained and the adenomas from two of these patients showed a heterogeneous mutation pattern in the APC gene. All detected mutations are predicted to result in a truncated APC protein. Genetic alterations in the mutation cluster region of the APC gene occurred significantly more frequently in large adenomas and showed a trend towards an increase in villous adenomas and adenomas with moderate and severe dysplasia. It is concluded that an increased proportion of APC gene mutations were found in the adenomas showing clinicopathological features associated with increased proliferation but not with characteristics of malignant potential. The results suggest that in a significant proportion of adenomas, other (genetic) alterations are involved in early tumourigenesis.

Adenoma↗

PubMed2003

Autosomal dominant external ophthalmoplegia and bipolar affective disorder associated with a mutation in the ANT1 gene.

The authors report on a family with dominantly inherited progressive external ophthalmoplegia and a diagnostic and statistical manual (fourth revised edition) diagnosis of bipolar psychiatric disorder in several members. Skeletal muscle biopsy from the proposita showed decreased cytochrome c oxidase staining, several ragged-red fibers, and multiple mtDNA deletions. The authors identified a missense mutation (leucine 98-->proline) in the adenine nucleotide translocator 1 gene. The presence of bipolar affective disorder expands the phenotype of adenine nucleotide translocator 1 allelic variants.

Adenine Nucleotide Translocator 1↗

PubMed1998

The human hepatocyte growth factor receptor gene: complete structural organization and promoter characterization.

The hepatocyte growth factor (HGF) receptor is a tyrosine kinase transmembrane protein encoded by the c-met proto-oncogene. Here, we have isolated and characterized human genomic DNA clones containing the entire coding sequence of the HGF receptor. The gene spans approx. 120kb in length and consists of 21 exons interrupted by 20 introns. Two alternative splice sites corresponding to known mRNA transcripts are located at exon 10 and exon 14, respectively. Structural analysis of the 5'-regulatory region reveals that the c-met promoter lacks TATA or CAAT elements but has an extremely high G-C content and multiple Sp1 binding sites. By transfection of a series of chimeric reporter constructs containing a variable region of the c-met promoter and the coding region for chloramphenicol acetyltransferase, we have identified two positive, and one negative regulatory elements that dictate c-met transcription in renal epithelial mIMCD-3 cells at nucleotide positions -2615 to -1621, -223 to -68, and -1621 to -1093, respectively. Moreover, deletion and mutation of the multiple Sp1 sites in the c-met promoter region markedly reduced c-met promoter activity in mIMCD-3 cells, suggesting that the Sp1 sites are essential for establishing the constitutive expression of the c-met gene. These data provide fundamental information on HGF receptor gene organization, as well as on the genomic origin of different receptor isoforms, and should facilitate further studies on the transcriptional regulation of its expression.

Alternative Splicing↗

PubMed2026

Elucidating the Role of SET as a Key Contributor to Neurodevelopmental Disability Within the 9q34.11 Deletion Syndrome Interval.

The 9q34.11 chromosomal region contains multiple neurodevelopmental genes involved in synaptic transmission, axonal structure and neuronal maturation. Pathogenic microdeletions, duplications and single nucleotide variants in numerous genes were previously linked with neurodevelopmental disorders (NDDs). Amongst them, SET has recently been implicated in a rare NDD with speech delay and facial dysmorphism. This study reports a female with a heterozygous de novo deletion impacting SET but not other NDD-associated genes at 9q34.11. The proband was initially diagnosed with atypical Rett syndrome with overlapping clinical features of SET haploinsufficiency. The deletion was confirmed using microarray and long-read sequencing. Subsequent quantitative proteomic evaluation identified a significant decrease of SET protein in patient-derived fibroblasts compared to control lines. This study provides insights into the proband's clinical course over their 28 year diagnostic odyssey, and emphasises the benefits of early speech therapy interventions. The proband had no functional speech, but regained the capacity to meaningfully communicate and articulate a limited vocabulary in adulthood, concordant with other reported non-paediatric cases of SET-NDD. This study expands current knowledge on the genotypic and phenotypic spectra of SET-NDD, and pinpoints a smaller 9q34.11 critical region excluding upstream NDD-associated genes, STXBP1 and SPTAN1, implicating SET as a significant NDD-associated gene.

Humans↗

PubMed2001

Diseases caused by nuclear genes affecting mtDNA stability.

Diseases caused by nuclear genes that affect mitochondrial DNA (mtDNA) stability are an interesting group of mitochondrial disorders, involving both cellular genomes. In these disorders, a primary nuclear gene defect causes secondary mtDNA loss or deletion formation, which leads to tissue dysfunction. Therefore, the diseases clinically resemble those caused by mtDNA mutations, but follow a Mendelian inheritance pattern. Several clinical entities associated with multiple mtDNA deletions have been characterized, the most frequently described being autosomal dominant progressive external ophthalmoplegia (adPEO). MtDNA depletion syndrome (MDS) is a severe disease of childhood, in which tissue-specific loss of mtDNA is seen. Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) patients may have multiple mtDNA deletions and/or mtDNA depletion. Recent reports of thymidine phosphorylase mutations in MNGIE and adenine nucleotide translocator mutations in adPEO have given new insights into the mechanisms of mtDNA maintenance in mammals. The common mechanism underlying both of these gene defects could be disturbed mitochondrial nucleoside pools, the building blocks of mtDNA. Future studies on MNGIE and adPEO pathogenesis, and identification of additional gene defects in adPEO and MDS will provide further understanding about the mammalian mtDNA maintenance and the crosstalk between the nuclear and mitochondrial genomes.

Blotting, Southern↗