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PubMed1992

Identification and characterization of a novel insertion sequence, IS1106, downstream of the porA gene in B15 Neisseria meningitidis.

Examination of Neisseria meningitidis strains associated with endemic meningococcal disease demonstrated differences in the number of copies of a repetitive sequence. Characterization of a copy of this repetitive sequence present in B15 strains has revealed the presence of a novel insertion sequence (IS1106) located within a complex repetitive region downstream of the gene for the major surface antigen (porA). IS1106 has a length of 1137 bp and is flanked by 36bp inverted repeats. Two open reading frames (ORF1 and ORF2) are present in opposite strands in codon-codon register with ORF2 entirely located within ORF1. The predicted protein from ORF1 demonstrates homology with the 5A protein of IS5 (Kroger and Hobom, 1982). Strains from two independent outbreaks of B15 meningococcal disease in the UK were found to contain the same genomic deletion removing a copy of IS1106 downstream of the porA gene.

Alleles↗

PubMed1995

Genetic analysis of three loci homologous to human G9a: evidence for linkage of a class III gene with the chicken MHC.

The cDNA clones of two newly discovered genes in the class III region of the human major histocompatibility complex (MHC) were hybridized to chicken DNA. One of these cDNA clones (pG9a-4C7), which detects the single-copy human G9a (BAT8) gene, gave a repeatable restriction pattern. This heterologous cDNA clone was used to detect and map three different PstI restriction fragment length polymorphisms among the two internationally recognized chicken reference populations. Two of the loci were unlinked to previously mapped markers, but one polymorphism cosegregated with the EaB locus in the Compton mapping population. These results provide evidence that some genes of the mammalian class III region, such as G9a, may be linked to the MHC in chickens.

Animals↗

PubMed1991

Partial conservation of the 5' ndhE-psaC-ndhD 3' gene arrangement of chloroplasts in the cyanobacterium Synechocystis sp. PCC 6803: implications for NDH-D function in cyanobacteria and chloroplasts.

The psaC gene, which encodes the 8.9 kDa iron-sulfur containing subunit of Photosystem I, has been sequenced from Synechocystis sp. PCC 6803 and shows greater similarity to reported plant sequences than other cyanobacterial psaC sequences. The deduced amino acid sequence of the protein encoded by the Synechocystis psaC gene is identical to the tobacco PSA-C sequence. In plants psaC is located in the small single-copy region of the chloroplast genome between two genes (designated ndhE and ndhD) with similarity to genes encoding subunits of the mitochondrial NADH Dehydrogenase Complex I. The 5' ndhE-psaC-ndhD3' gene arrangement of higher plants is only partially conserved in Synechocystis. An open reading frame (ORF) upstream of the Synechocystis psaC gene has 85% identity to the tobacco ndhE gene. Downstream of psaC there is a 273 bp ORF with 48% identity to the 5' portion of the tobacco ndhD gene (1527 bp). psaC, ndhE and the region of similarity to ndhD are present in a single copy in the Synechocystis genome. Part of the wheat ndhD gene was sequenced and used as a probe for the presence of the 3' portion of the ndhD gene. The wheat ndhD probe did not hybridize to Synechocystis or Anabaena sp. PCC 7120 genomic DNA, but did hybridize to Oenothera chloroplast DNA. These results indicate the complete ndhD gene is absent in two cyanobacteria, and raises the question of what role, if any, the ndhD gene product plays in the facultative heterotroph Synechocystis sp. PCC 6803.

Amino Acid Sequence↗

PubMed2001

The Drosophila dorsoventral determinant PIPE contains ten copies of a variable domain homologous to mammalian heparan sulfate 2-sulfotransferase.

In Drosophila, the gene PIPE is expressed in follicle cells, the somatic cells that surround the forming egg during maturation, specifically on one side of the egg chamber. This asymmetry establishes the dorsoventral axis of the future embryo. Through the action of PIPE, the ligand SPATZLE, that is located in the perivitelline fluid of the embryo, is activated ventrally. This signal activates TOLL, a membrane-bound receptor. According to present knowledge, PIPE encodes two different transcripts, one of which restored ventral pattern elements to embryos when introduced into mutant pipe females. Here we show that PIPE is far more complex than previously reported. It encodes not two, but at least ten different transcripts, two of which are localized to ventral follicle cells. The transcripts contain one of ten copies of a variable domain, all homologous to heparan sulfate 2-sulfotransferase, an enzyme known to modify heparan sulfate proteoglycans, which are molecules that can bind ligands. The complex gene structure of PIPE thus evolved by duplications of one exon, a strategy used by genes of the immunoglobulin superfamily to generate molecular diversity. We show that PIPE transcripts can be eliminated by RNAi, although in this method double-stranded RNA is injected in embryos, while PIPE transcripts appear in the adult ovary. Our data suggest that at least two different PIPE transcripts redundantly provide the ventralizing PIPE function. 3' of PIPE we identified an enhancer element that drives a lacZ reporter gene specifically in ventral follicle cells. Since PIPE transcripts are found in salivary glands, and since expression of salivary gland genes is dependent on signaling molecules, we speculate that PIPE became localized to ventral follicle cells by a preexisting control system after acquiring a follicle cell enhancer.

Alternative Splicing↗

PubMed2005

The Arabidopsis thaliana SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASES1 and 2 control male sporogenesis.

The Arabidopsis thaliana SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) family of plasma membrane receptors consists of five closely related members. The SERK1 and SERK2 genes show a complex expression pattern throughout development. Both are expressed in anther primordia up to the second parietal division. After this point, expression ceases in the sporocytes and is continued in the tapetum and middle layer precursors. Single knockout mutants of SERK1 and SERK2 show no obvious phenotypes. Double mutants of SERK1 and SERK2 are completely male sterile due to a failure in tapetum specification. Fertility can be restored by a single copy of either gene. The SERK1 and SERK2 proteins can form homodimers or heterodimers in vivo, suggesting they are interchangeable in the SERK1/SERK2 signaling complex.

Alleles↗

PubMed1994

The glucose-regulated proteins (GRP78 and GRP94): functions, gene regulation, and applications.

The knowledge of GRPs as molecular chaperones is rapidly evolving. It is anticipated that the GRPs will make special contributions in the areas of basic cell biology, biotechnology, and cancer biology. In particular, they may play a role as the prototype of a class of genes that are regulated by signal transduction pathways originating in the ER and traveling to the nucleus. GRP78 and GRP94 function as molecular chaperones and can bind to malfolded proteins and unassembled complexes. They are induced in response to stress, but once the stress is removed the GRPs are posttranscriptionally modified into biologically inactive forms. The promoters of the grp genes are highly conserved, with several CCAAT-like motifs and GC-rich regions. The high level of redundancy that exists in the mammalian grp promoters may act to ensure that the expression of the genes, both of which are single copy, is unlikely to be significantly lowered in the event of mutation. These genes are thought to be controlled by several transcription factors whose complex interactions with the grp promoters allow variable patterns of grp induction. The promoters of the grp genes constitutively express their gene products, and their promoter activities can be further enhanced in cellular environments of low glucose or oxygen. The grp78 promoter is known to retain its strong activity in differentiated and undifferentiated tissues. These features make it an attractive alternative to viral promoters for use in gene therapy. Gene therapy may also be useful in treating cancer in some cases, especially solid tumors. In these instances, GRP levels are already likely to be quite high. These high levels of GRPs may inhibit the efficacy of several anti-cancer treatments. Suppression of GRP induction, perhaps by anti-sense or ribozyme technology, may prove to be useful in conjunction with anti-cancer drugs to treat tumors.

Animals↗

PubMed2006

Seventh international meeting on single nucleotide polymorphism and complex genome analysis: 'ever bigger scans and an increasingly variable genome'.

In September 2005, the seventh international meeting on single nucleotide polymorphism (SNP) and complex genome analysis was held in Hinckley, near Leicester, UK and the meeting was organised by Anthony Brookes, Stephen Chanock, Ivo Gut, Alec Jeffreys and Pui-Yan Kwok. Similar to prior meetings, the 3-day meeting focused on new trends and methods in the analysis of SNPs and complex human disease. A substantial portion of the meeting was devoted to preliminary analyses of data emerging from the International HapMap Consortium and addressed key issues in patterns of recombination, linkage disequilibrium and population genetics. Of great interest were the sessions that addressed SNP analysis in other species and the emerging field of copy number variation. Overall, there have been a number of recent advances in genomics that promise to accelerate the pace of dissecting the genetic basis of many complex diseases in humans-and perhaps in other species.

Evolution, Molecular↗

PubMed1991

Cloned single copy DNA sequences of Mycobacterium tuberculosis as DNA probes.

A recombinant genomic clone was isolated from a lambda gt 11 library of M. tuberculosis on the basis of lack of hybridization with M. avium and M. kansasi. The specificity and sensitivity of M. tb DNA probes, 2.5 and 2.3 kb in size, were assessed by Southern blot and dot blot hybridization. These did not cross hybridize to DNA of mycobacteria other than members of M. tb complex, nor with DNA of non mycobacterial origin. Sensitivity was determined to be 200 pg which is equivalent to 10(4) bacilli. Genomic Southern hybridization indicated single copy nature of the probes.

Cloning, Molecular↗

PubMed1987

Structural analysis of two genes encoding divergent forms of yeast cytochrome c oxidase subunit V.

In Saccharomyces cerevisiae, subunit V of the inner mitochondrial membrane protein complex cytochrome c oxidase is encoded by two nonidentical genes, COX5a and COX5b. Both genes are present as single copies in S. cerevisiae and in several other Saccharomyces species. Nucleotide sequencing studies with the S. cerevisiae COX5 genes reveal that they encode proteins of 153 and 151 amino acids, respectively. Overall, the coding sequences of COX5a and COX5b have nucleotide and protein homologies of 67 and 66%, respectively. They are saturated for nucleotide substitutions that result in a synonomous codon, indicating a long divergence time between these two genes. Nucleotide sequences flanking the COX5a and COX5b coding regions exhibit no significant homology. The COX5a protein, pre-subunit Va, contains a 20-amino-acid leader peptide, whereas the COX5b protein, pre-subunit Vb, contains a 17-amino-acid leader peptide. These two leader peptides exhibit only 45% homology in the primary sequence, but have similar predicted secondary structures. By analyzing the RNA transcripts from both genes we have found that COX5a is a contiguous gene but that COX5b contains an intron. Surprisingly, the COX5b intron interrupts the AUG codon that initiates translation of the pre-subunit Vb polypeptide and contains a 5' donor splice sequence that differs from that normally found in yeast introns.

Amino Acid Sequence↗

PubMed2000

Identification of two novel TAF subunits of the yeast Saccharomyces cerevisiae TFIID complex.

Using a combination of ion exchange and immunoaffinity chromatography we have purified the general transcription initiation factor TFIID to near homogeneity from Saccharomyces cerevisiae. Yeast TFIID is composed of TBP, the TATA box binding protein, and 14 distinct TBP-associated factors (TAFs), which range in size from 17 to 150 kDa. Twelve of the TAF subunits have been previously identified, but two, TAF48p and TAF65p, are novel. TAF48p exhibits significant sequence similarity to the conserved C-terminal region of Drosophila TAF110p, human TAF130p, and human TAF105p and is encoded by a previously identified gene MPT1. TAF65p shows no significant sequence homology to any previously identified TAFp. The genes encoding TAF48p and TAF65p are single copy and essential for normal yeast cell growth. Furthermore, neither TAF48p nor TAF65p are associated with the histone acetylase Spt-Ada-Gcn5 complex or other non-TFIID TBF.TAF complexes. The significance of these results in terms of TFIID structure, function, and organization is discussed.

Amino Acid Sequence↗

PubMed2003

Building and breaking bridges between sister chromatids.

Eukaryotic chromosomes undergo dramatic changes and movements during mitosis. These include the individualization and compaction of the two copies of replicated chromosomes (the sister chromatids) and their subsequent segregation to the daughter cells. Two multisubunit protein complexes termed 'cohesin' and 'condensin', both composed of SMC (Structural Maintenance of Chromosomes) and kleisin subunits, have emerged as crucial players in these processes. Cohesin is required for holding sister chromatids together whereas condensin, together with topoisomerase II, has an important role in organizing individual axes of sister chromatids prior to their segregation during anaphase. SMC and kleisin complexes also regulate the compaction and segregation of bacterial nucleoids. New research suggests that these ancient regulators of chromosome structure might function as topological devices that trap chromosomal DNA between 50 nm long coiled coils.

Adenosine Triphosphatases↗

PubMed2001

Characterization of chromosomal abnormalities in uroepithelial carcinomas by G-banding, spectral karyotyping and FISH analysis.

Chromosome analysis by G-banding, spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH) was performed on 24 short-term cultured transitional cell bladder carcinomas and 5 cell lines established from bladder carcinomas. Except for one tumor with an apparently normal chromosomal constitution, clonal chromosome abnormalities were detected in all examined cases by the combined approach. The application of SKY and FISH techniques improved the karyotypic descriptions, originally based on G-banding only, by identifying 32 additional numerical changes, by establishing the chromosomal origin of 27 markers and 2 ring chromosomes, by redefining 53 aberrations and by detecting 15 hidden chromosomal rearrangements. No recurrent translocation, however, was detected. The most prominent karyotypic feature was thus the occurrence of deletions and losses of whole chromosome copies indicating the importance of tumor suppressor genes in transitional cell carcinoma pathogenesis. Invasive carcinomas were karyotypically more complex than were low grade superficial tumors. Specific losses of material from chromosome 9 and from chromosome arms 11p and 8p, and gains of 8q and 1q seem to be early changes appearing in superficial tumors, whereas losses from 4p and 17p and the formation of an isochromosome for 5p were associated with more aggressive tumor phenotypes.

Aged↗

PubMed1995

DNA cleavage at two recognition sites by the SfiI restriction endonuclease: salt dependence of cis and trans interactions between distant DNA sites.

At low ionic strength, the SfiI restriction enzyme cleaved at similar rates both supercoiled and linear DNA with two SfiI sites and linear DNA with one SfiI site. For the substrates with two sites, the majority of the DNA was converted directly to products cut at both sites; the enzyme appears to bind to two sites before catalyzing its reactions, looping out the intervening DNA. At high ionic strength, linear DNA with one SfiI site was not cut at all, linear DNA with two sites was cleaved slowly while supercoiled DNA with two sites was cleaved rapidly, though only half of the DNA with two sites was cut at both sites; the DNA that had been cut at one site was not cleaved again at the remaining site. The singly cut product must therefore have been generated by a reaction incorporating both sites. All DNA cleavage reactions by SfiI thus involve the tetrameric enzyme bound to two copies of its recognition sequence, but weakened DNA-protein interactions at high ionic strength can cause this complex to dissociate before cleaving both sites. Intramolecular interactions between distant DNA sites are generally thought to be enhanced by supercoiling and to be more stable than intermolecular interactions. The preference of SfiI at high ionic strength for substrates with two sites over substrates with one site and, in the former case, for supercoiled over linear DNA, validates this view. At low ionic strength, the similar rates with the different substrates may be due to rate-limiting product dissociation.

Base Sequence↗

PubMed1999

The F plasmid centromere, sopC, is required for full repression of the sopAB operon.

The SopB protein of the F plasmid has a dual role in the partition of F plasmid copies to daughter cells prior to division. It binds to the sopC centromere site to form the partition complex needed for stabilizing the plasmid, and it interacts with SopA to repress transcription of the sopAB operon, thus preventing the destabilization that results from excess SopB. We have isolated sop mutants by screening for unstable inheritance of mutagenized mini-F DNA. Four of the mutants resulted from different missense mutations in sopB. All four were deficient, to varying degrees, in autoregulation of Sop protein synthesis. The mutant proteins showed diminished capacity for reducing the linking number of mini-F and for destabilizing a plasmid carrying sopC, indicating that reduced ability to form a normal complex with sopC might underlie the autoregulation defect. Repression of the transcription of a sop promoter- lacZ fusion by SopA and SopB was strongly enhanced in the presence of sopC, in cis or in trans, and the enhancement was reduced or nullified when wild-type sopB was replaced by the mutant sopB alleles. A single 43 bp unit of sopC was almost as effective as sopC itself in enhancing repression. The results show that sopC is necessary for full repression of the sop promoter. They thus indicate a previously unsuspected role for this centromere site, and suggest that autoregulation and partition might normally be coordinated processes.

Bacterial Proteins↗

PubMed1995

Physical and gene organization of mitochondrial DNA from the fertile cytoplasm of sugarbeet (Beta vulgaris L.).

We have constructed a complete physical map of the mitochondrial genome from the male-fertile cytoplasm of sugarbeet. The entire sequence complexity can be represented on a single circular master chromosome of 358 kb. This master chromosome contains three copies of one recombinationally active repeat sequence, with two copies in direct orientation and the other in inverted orientation. The positions of the rRNA genes and of 23 polypeptide genes, determined by filter hybridization, are scattered throughout the genome, with triplicate rrn26 genes located partially or entirely within the recombination-repeat elements. Three ribosomal-protein genes (rps1A, rps14 and rps19) were found to be absent from sugarbeet mtDNA. Our results also reveal that at least six regions homologous with cDNA are dispersed in the mitochondrial genome.

Chromosome Mapping↗

PubMed2004

Hypopituitarism as a consequence of traumatic brain injury (TBI) and its possible relation with cognitive disabilities and mental distress.

Recent studies have demonstrated that hypopituitarism, in particular GH deficiency, is common among survivors of traumatic brain injury (TBI) tested several months or yr following head trauma. We present the results of endocrine, neurological, neuropsychological and psychiatric evaluation in a group of 67 patients who suffered TBI at least one yr ago. Our study shows that decreased endocrine function is either restricted to one or more anterior pituitary hormones and is present in 34% of patients with any pituitary hormone deficit, while multiple pituitary hormone deficiencies are found in 10% of patients. GH/IGF-I axis was evaluated by GHRH+GHRP-6 test and IGF-I measurement. Severe GHD is the most frequent deficiency present in 15% of TBI patients. Gonadotrophin deficiency was present in 9% of patients with TBI, while thyrotroph and corticotroph function seemed more refractory to impairment. Patients with moderate-to-severe trauma are not necessarily more likely to have hypopituitarism than those with mild injury. Neuropsychological testing revealed a significant positive correlation of peak GH levels after GHRH+GHJRP-6 test with verbal learning and verbal short term memory (RAVLT total score p = 0.06, immediate free recall p = 0.02 and delayed free recall p = 0.04). Verbal and visual memory was significantly lower in elderly patients and in males. Visoconstructional abilities (RCF copy) were significantly lower in the elderly (p < 0.01) and undereducated (p = 0.02). Visual memory (free recall of complex figure after 30 min) significantly correlated with lower IGF-I levels (p = 0.01). Gonadotrophins and testosterone correlated significantly with visoconstructional abilities. Simple and complex conceptual tracking (TMT A and B) was significantly more impaired in older TBI patients (p < 0.01) and with longer time from trauma (TMT B only, p = 0.03). The psychiatric evaluation by using two different scales showed depression, phobic anxiety and psychoticism to be more prominent in the TBI group. Paranoid ideation and somatization negatively correlated with the peak GH responses to GHRH+GHRP-6 test (p = 0.04 and p = 0.03, respectively). Depression scale showed that nearly half of patients suffered from mild to moderate depression. The benefits of hormone replacement therapy on cognitive functioning and mental distress in TBI patients are eagerly awaited.

Adult↗

PubMedDate not supplied

Building a secretory apparatus: role of ARF1/COPI in Golgi biogenesis and maintenance.

The secretory apparatus within all eukaryotic cells comprises a dynamic membrane system with bidirectional membrane transport pathways and overlapping compartmental boundaries. Membrane traffic and organelle biogenesis/maintenance are fundamentally linked within this system, with perturbations in membrane traffic quickly leading to changes in organelle structure and identity. Dissection of the molecular basis of these properties in yeast and mammalian cells has revealed a crucial role for the cytoplasmic protein complex ARF1/COPI, which undergoes regulated assembly and disassembly with membranes. ARF1/COPI appears to be involved in the formation and maintenance of the Golgi complex, which is the receiving and delivery station for all secretory traffic. ARF1-GTP, through assembly of COPI to membranes and, possibly, through activation of PLD, is likely to promote the formation and maturation of pre-Golgi intermediates into Golgi elements, whereas ARF-GDP causes COPI dissociation and stimulates the formation of retrograde transport structures that recycle Golgi membrane back to the ER. These processes are appear to underlie the coupling of organelle biogenesis and membrane trafficking within cells, allowing the size and shape of secretory organelles to be altered in response to changing cellular needs. Future work needs to address how the activation and localization of ARF1/COPI to membranes as well as other related factors are temporally and spatially regulated, and by what mechanism they transform membrane shape and dynamics to facilitate protein transport and compartmental functioning.

ADP-Ribosylation Factor 1↗

PubMed1992

Complementary DNA sequences of two 14.5 kDa subunits of NADH:ubiquinone oxidoreductase from bovine heart mitochondria. Completion of the primary structure of the complex?

The amino acid sequences of two nuclear-encoded subunits of complex I from bovine heart mitochondria have been determined. Both proteins have an apparent molecular weight of 14.5 kDa and their N-alpha-amino groups are acetylated. They are known as subunits B14.5a and B14.5b. Neither protein is evidently related to any known protein and their functions are obscure. A total of 34 nuclear-encoded subunits of bovine complex I have now been sequenced and it is thought that the primary structure of the complex is now complete, although with such a complicated structure it is difficult to be certain that there are no other subunits remaining to be sequenced. Seven additional hydrophobic subunits of the enzyme are encoded in mitochondrial DNA, and therefore bovine heart complex I is an assembly of about 41 different proteins. If it is assumed that there is one copy of each protein in the assembly, these polypeptides contain 7,955 amino acids in their sequences, more than are found in the Escherichia coli ribosome, which contains 7,336 amino acids in its 32 polypeptides.

Amino Acid Sequence↗