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Comparative distribution of NURR1 and NUR77 nuclear receptors in the mouse central nervous system.

NURR1 and NUR77 are members of the nuclear receptor superfamily of transcription factors. Both proteins can interact with common enhancer elements to regulate target gene expression. In order to establish whether both transcription factors are likely to regulate overlapping genes, we have used an in situ hybridization approach to relate the constitutive expression pattern of these mRNAs with functionally defined regions of the adult mouse brain. By Western analysis, NURR1 mRNA expressed by brain cells appeared to be translated. Here we show that both transcripts display a differential but partially overlapping pattern of expression within the central nervous system (CNS). The expression of NURR1 is more restricted than NUR77 and is localized predominantly in sensory neuronal structures associated with the limbic system and in the cerebellum. In contrast, the expression pattern of NUR77 is more widespread. Positively staining cells for NUR77 appear to overlap with NURR1-containing cells in the limbic system and cerebellum, suggesting overlapping roles for these proteins in mediating behavioral and cognitive function as well as equilibrium maintenance. However, the differential expression of NUR77 in motor areas of the cortex and basal ganglia suggest a selective role for this transcription factor in regulation of motor function at the constitutive level. Our data indicates that these nuclear receptors are likely to have both shared and independent gene regulatory roles in neuronal cells.

Age Factors↗

Effect of hepatitis C virus core shadow protein expressed in human hepatoma cell line on human gene expression profiles.

BACKGROUND AND AIMS: The hepatitis C virus (HCV) C region has been reported to have overlapping genes or regions, and may encode a core shadow protein that has a role in HCV self-replication, pathogenesis and carcinogenesis. The aim of this study was to identify the effect of HCV core shadow protein expressed in a human hepatoma (Huh-7) cell line on human gene expression profiles. METHODS: Recombinants for expression of HCV genotype 1b core shadow protein and genotype 1b core protein were constructed, and an Huh-7 cell line was established that could express the shadow protein and the core protein constitutively. Affymetrix human gene chip, HG-U133 A and B microarray analysis and semiquantitative RT-PCR were employed to identify the expression profiles of two kinds of core proteins in the Huh-7 cell line. RESULTS: The microarray analysis showed that the core shadow protein caused expression of more genes to be up/down-regulation than the core protein, including signal transduction, protease activity, molecular transport and, particularly, immune responses genes. Surprisingly, the core shadow protein could increase/decrease expression of apoptosis and anti-apoptosis genes simultaneously. The expression profiles of three up-regulated genes were confirmed by semiquantitative RT-PCR, with results similar to the microarray analysis. CONCLUSIONS: Hepatitis C virus core shadow protein may play an important role in inhibiting or stimulating host cells apoptosis processing and carcinogenesis, which is useful for the understanding of HCV core shadow protein biological functions in vivo and in vitro.

Blotting, Western↗

Comparison of polymerase chain reaction systems for detection of different cdt genes in Escherichia coli strains.

AIMS: Cytolethal distending toxins (CDT) are tripartite toxins encoded by three adjacent or overlapping genes (cdtA, cdtB, cdtC) and found in multiple pathogens. The present knowledge regarding heterogeneity of cdt genes and our previous study revealed that the available polymerase chain reaction (PCR) systems lack adequate specificity. The detection of various cdt genes present in Escherichia coli strains, from different geographical regions demands further assays for wide-range coverage. On the basis of these observations, we were prompted to undertake the present study; hence the specificity of existing PCR systems was addressed using E. coli prototype strains with known cdt gene sequences. METHODS AND RESULTS: A multiplex PCR designed for the detection of E. coli cdt genes was found to be sensitive and specific enough for initial screening. However, for subtyping, the PCR systems yielded nonspecific products upon amplification. These primers are usually designed for sequences of the cdtB locus (the most conserved region of the gene), and since CDT-producing E. coli strains carry different cdt genes, none of the systems are really type specific. Furthermore, PCR systems with type-specific primers for other regions of the gene, i.e. ORF A or ORF C are found to be strain specific and their applications in different geographical regions have limitations. CONCLUSIONS: In conclusion, based on our observations, using these available primers, it seems that the existing PCR systems are not sufficiently accurate to differentiate between different types of cdt genes. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained from this study revealed that so-far reported PCR systems are short in specificity. These PCR protocols were not found to be specific enough to detect various cdt genes and have a limited range of application. Moreover, due to similarities in cdt genes the cross-reaction between different sets of primers exists. Hence for epidemiological studies, some additional PCR protocols are required for screening clinical isolates for cdt genes.

Bacterial Toxins↗

The plastid genome of the cryptophyte alga, Guillardia theta: complete sequence and conserved synteny groups confirm its common ancestry with red algae.

The plastid genome of the cryptophyte alga Guillardia theta (121,524 bp) has been completely sequenced. The genome is 33% G+C and contains a short, nonidentical inverted repeat (4.9 kb) encoding the two rRNA cistrons. The large and small single-copy regions are 96.3 and 15.4 kb, respectively. Forty-six genes encoding proteins for photosynthesis, 5 genes for biosynthetic function, 5 genes involved in replication and division, 30 tRNA genes, 44 ribosomal protein genes (26 large subunit and 18 small subunit), 3 translation factors, 8 genes encoding components of the transcriptional machinery including 3 ycfs (hypothetical chloroplast frames), and 26 additional ycfs have been identified. There are eight ORFs larger than 50 amino acids, 3 of which have homologues on the plastid genome of the rhodophyte, Porphyra purpurea (Reith and Munholland 1995) and/or the Synechocystis genome (Kaneko et al. 1996) and can be designated new ycfs. Intergenic spacers are very short, no introns have been detected, and several genes overlap, all resulting in a very compact genome. In addition, large clusters of genes (such as those for the ribosomal proteins) are organized into single transcriptional units (Wang et al. 1997), again resulting in an economically organized genome. The cryptophyte plastid genome is almost completely comprised of clusters of genes that are found on the rhodophyte Porphyra purpurea, confirming its common ancestry with red algae. Furthermore, recombination events involving both tRNA genes and the rRNA cistrons appear to have been responsible for the structure of the cryptophyte plastid genome, including the formation of the inverted repeat.

Cell Division↗

Exact localization of the familial dysbetalipoproteinemia associated HpaI restriction site in the promoter region of the APOC1 gene.

An HpaI restriction fragment length polymorphism (RFLP) in the APOE-C1-C2 gene cluster on chromosome 19 is strongly associated with familial dysbetalipoproteinemia (type III hyperlipoproteinemia). Recently we localized this polymorphic HpaI site between the APOE and APOC1 genes. In the present paper we show by molecular cloning and sequencing that the polymorphic HpaI site is located 317 bp upstream of the transcription initiation site of the APOC1 gene. Overlapping cosmid clones allowed the construction of a detailed restriction map of the gene cluster, showing the APOC2 gene to be located 15 kb downstream of the APOC1 pseudogene.

Apolipoprotein C-I↗

Molecular cloning and expression of NlaIII restriction-modification system in E. coli.

The NlaIII restriction enzyme isolated from Neisseria lactamica recognizes the sequence 5'-CATG-3', cleaving after the G to generate a four base 3' overhang. The NlaIII methylase and a portion of the NlaIII endonuclease gene were cloned into E. coli by the methylase selection method, and the remaining portion of the NlaIII endonuclease gene was cloned by inverse PCR. The nucleotide sequence of the endonuclease gene and the methylase gene were determined. The NlaIII endonuclease gene is 693 bp, encoding a protein with predicted molecular weight of 26487. The NlaIII methylase gene was identical with that previously reported [Labbe, D., Joltke, H.J. and Lau, P.C. (1990) Cloning and characterization of two tandemly arranged DNA methyltransferse genes of Neisseria lactamica: an adenine-specific M.NlaIII and a cytosine-type methylase. Mol. Gen. Genet. 224, 101-110]. The endonuclease and methylase genes overlap by four bases and are transcribed in the same orientation. The endonuclease gene was cloned into an improved T7 vector, and a high level of NlaIII endonuclease expression was achieved in E. coli.

Amino Acid Sequence↗

Enteroaggregative escherichia coli virulence factors in traveler's diarrhea strains.

Enteroaggregative Escherichia coli (EAEC) is associated with diarrhea in Spanish travelers to developing countries. In this study, the polymerase chain reaction was used to test EAEC isolates for genes encoding putative virulence factors, including EAEC adhesins, the plasmid-encoded toxin (Pet), a heat-stable enterotoxin (EAST), and Shigella enterotoxins 1 and 2 (ShET1 and ShET2). Findings included a low prevalence of genes for Pet (4.3%), ShET2 (4.3%), and the adherence factor AAF/II (8.7%). The overlapping genes encoding the ShET1 and the Pic mucinase were present in most EAEC strains tested (56.5%); however, some strains that carried this locus did not produce both proteins, as determined by Western immunoblot. Surprisingly, ShET1 and ShET2 genes were also found in other E. coli pathotypes, as was the EAST toxin locus. These findings underscore the heterogeneity of EAEC strains and suggest that the ShET1 may be an important virulence factor in traveler's diarrhea.

Adhesins, Escherichia coli↗

Construction and properties of a ribosome-binding site mutation in gene E of phi X174 bacteriophage.

Oligodeoxyribonucleotide mutagenesis has been used to produce a G----A mutation at nucleotide 557 of the phi X174 genome. This changes the ribosome-binding sequence GAGG of gene E to GAAG without affecting the amino acid, glutamine, encoded by the overlapping gene D. The phi X174rb(E)557 mutant does not lyse infected Escherichia coli C and therefore results in the accumulation of a large number intracellular mature phage particles. Thus, the mutation inactivates production of the gene E lytic product, presumably by blocking translation of gene E, without affecting other phage functions.

Bacteriophage phi X 174↗

Gene K of bacteriophage phi X174 codes for a protein which affects the burst size of phage production.

Site-directed mutagenesis has been used to produce a T----A change at nucleotide 70 of phi X174 genome. This generates an am codon, TAG, in the gene K reading frame without affecting the amino acid, leucine, encoded by the overlapping gene A. The gene K mutant produces small plaques on su- hosts. It has an identical latent period, but a more reduced burst size than that of the wild-type phi X174. The reduced burst size in the gene K mutant suggests that the gene K protein, although not essential, has a role in increasing infectivity by increasing the burst size three- to sixfold.

Bacteriophage phi X 174↗

Natural antisense transcripts.

The sequencing of whole genomes and the subsequent annotation of cDNAs revealed that about 20% of human and mouse genes overlap resulting in potential pairs of sense and antisense transcripts. An increasing number of experimentally identified antisense transcripts concur with this predication. Characterization of overlapping transcripts in various species indicates that this form of RNA-mediated gene regulation represents a widespread phenomenon. However, the physiological relevance of natural antisense transcripts remains obscure. Genomic studies suggest that duplex formation between sense and antisense is required for biological function. Antisense transcripts play an established role in imprinting and X-chromosome inactivation and genomic rearrangements as observed in B and T leukocytes. Only a relatively small percentage of the predicted antisense transcripts are related to these biological phenomena that are also related to mono-allelic expression. Consequently, there are at least two categories of natural antisense transcripts that show significant differences with regard to their biological function as well as the potential mechanisms involved.

Animals↗

[Interactive pathway of ARF-mdm2-p53].

The inhibitor of cyclin-dependent kinase 4-p16INK4 alpha and its alterative reading frame (ARF)-INK4/ARF gene locates at CDKN2A locus of human chromosome 9p21. This locus encodes two overlapped genes: ARF gene and p16INK4 alpha. The amino acid sequences of two genes are completely different because they are encoded by alternative reading frames. ARF participates in the regulation of mdm2-p53 pathway by mdm2. Recent studies showed that ARF gene may play a role in tumorigenesis; the ARF gene promoter hypermethylation may be the principal mechanism in the inactivation of this gene. Here is a review of ARF-mdm2-p53 interacting pathway.

Alternative Splicing↗

Transmissible gastroenteritis coronavirus gene 7 is not essential but influences in vivo virus replication and virulence.

Transmissible gastroenteritis coronavirus (TGEV) contains eight overlapping genes that are expressed from a 3'-coterminal nested set of leader-containing mRNAs. To facilitate the genetic manipulation of the viral genome, genes were separated by duplication of transcription regulating sequences (TRSs) and introduction of unique restriction endonuclease sites at the 5' end of each gene using an infectious cDNA clone. The recombinant TGEV (rTGEV) replicated in cell culture with similar efficiency to the wild-type virus and stably maintained the modifications introduced into the genome. In contrast, the rTGEV replication level in the lungs and gut of infected piglets and virulence were significantly reduced. rTGEV in which gene 7 expression was abrogated (rTGEV-delta7) were recovered from cDNA constructs, indicating that TGEV gene 7 was a nonessential gene for virus replication. Interestingly, in vivo infections with rTGEV-delta7 showed an additional reduction in virus replication in the lung and gut, and in virulence, indicating that TGEV gene 7 influences virus pathogenesis.

Animals↗

Appropriate expression of filamentous phage f1 DNA replication genes II and X requires RNase E-dependent processing and separate mRNAs.

The products of in-frame overlapping genes II and X carried by the filamentous phage f1 genome are proteins with required but opposing functions in phage DNA replication. Their normal relative levels are important for continuous production of phage DNA without killing infected Escherichia coli hosts. Here we identify several factors responsible for determining the relative levels of pII and pX and that, if perturbed, alter the normal distribution of the phage DNA species in infected hosts. Translation of the two proteins is essentially relegated to separate mRNAs. The mRNAs encoding genes II and X are also differentially sensitive to cleavage dependent on rne, the gene encoding the only E. coli endo-RNase known to have a global role in mRNA stability. Whereas pII levels are limited at the level of mRNA stability, normal pX levels require transcription in sufficient amounts from the promoter for the smaller mRNA encoding only pX.

Base Sequence↗

HTLV-II transactivation is regulated by the overlapping tax/rex nonstructural genes.

The human T-cell leukemia virus (HTLV) types I and II have two nonstructural genes that are encoded in overlapping reading frames. One of these genes, known as tax, has been shown to encode a protein responsible for enhanced transcription (transactivation) from the viral long terminal repeats (LTRs). Genetic evidence indicates that the second nonstructural gene of HTLV-II, here designated rex, acts in trans to modulate tax gene-mediated transactivation in a concentration-dependent fashion. The rex gene may regulate the process of transactivation during the viral life cycle.

Base Sequence↗

Surf5: a gene in the tightly clustered mouse surfeit locus is highly conserved and transcribed divergently from the rpL7A (Surf3) gene.

The four previously characterized genes (Surf1 to 4) of the mouse Surfeit locus do not share any sequence homology, and the transcription of each gene alternates with respect to its neighbor(s). Adjacent Surfeit genes are separated by very small distances, and two of the genes overlap at their 3' ends. In this work we have further defined the Surfeit gene cluster by the isolation of Surf5, a fifth gene of the locus, and determination of its relationship to the other Surfeit genes. Surft5 does not share any sequence homology with the four cloned Surfeit genes. The transcription of Surf5 is divergent with respect to its neighbor the Surf3 gene, and the 5' ends of Surf5 and Surf3 are separated by only 159 bp, suggesting the presence of a second bidirectional promoter in the locus. The 3' end of Surf5 maps only 68 bp away from the processed 3' end of a pseudogene. The human and partial chicken Surf5 coding regions show greater than 95% identity, and a Caenorhabditis elegans homologue shows 38% identity and 56% similarity with the mouse Surf5 amino acid sequence. The 3.5-kb transcript of Surf5 encodes a small hydrophilic protein of 140 amino acid residues, which differs from the ribosomal protein L7a encoded by the Surf3 gene or the integral membrane protein encoded by the Surf4 gene. Subcellular fractionation located the Surf5 protein to the soluble fraction of the cytoplasm. The surfeit locus appears to represent a novel type of gene cluster in which the genes are unrelated by sequence or function; however, their organization may play a role in their gene expression.

Amino Acid Sequence↗

Functional analyses and identification of two arginine residues essential to the ATP-utilizing activity of the triple gene block protein 1 of bamboo mosaic potexvirus.

The TGBp1 of bamboo mosaic potexvirus (BaMV) is encoded by the first overlapping gene of the triple-gene-block (TGB), whose products are thought to play roles in virus movement between plant cells. This protein forms cytoplasmic inclusions associated with virus particles in the BaMV-infected tissues. It has been proposed that the inclusion is one of the active forms of TGBp1. To prove this idea, we purified the TGBp1 inclusions from both the BaMV-infected Chenopodium quinoa and Escherichia coli cells overexpressing this protein to test some of their biochemical activities. We found that the TGBp1 inclusions isolated from the infected plant leaves, but not from E. coli, possess the NTP-binding and NTPase activities. However, they lack the RNA-binding activity possessed by the soluble TGBp1. These results indicate that the TGBp1 proteins in the BaMV-infected tissues assume two different functional forms. Mutational analyses and competition experiments show that the two arginine residues, Arg-16 and Arg-21, essential to RNA binding, are also required for the ATP-utilizing activity of the soluble TGBp1. This indicates that a same-structure motif is required for the two functions of the soluble TGBp1. The location of the two arginine residues outside the seven conserved motifs of the NTP-utilizing superfamily I RNA helicases, to which TGBp1 belongs, suggests that an extra-structure motif, besides the seven conserved ones, is required for the NTP-utilizing activity of the TGBp1 protein of BaMV.

Acid Anhydride Hydrolases↗

Lambda transducing phages derived from a FinO- R100::lambda cointegrate plasmid: proteins encoded by the R100 replication/incompatibility region and the antibiotic resistance determinant.

Three lambda transducing phages have been isolated from pEDR20, an R100::lambda cointegrate plasmid in which the lambda insertion inactivated the R100 finO gene. Physical analysis of the three phages showed that the lambda is inserted at kilobase coordinate 81.3 of R100. All three phages carry different amounts of R100 DNA in the left arm of lambda. Each pahge contains ISlb, the mer genes and the region between coordinate 81.3 and 88.6; thus, all contain the genes necessary for R100 replication. One phage, VA lambda 73, contains the entire r-determination of R100 in addition to the above DNA. Five proteins coded by the region between 81.3 and 88.6 were detected. These had subunit molecular weights of 10,400; 12,200; 16,200; 19,600; and 38,300. The first was made constitutively and the other four only from a lambda promoter. Other constitutive proteins were one from the cml fus region with a molecular weight of 22,400 (cml) and two from the str sul region with molecular weights of 31,500 (str?) and 30,100 (sul?). Mercuric ion induced synthesis of at least 10 proteins. Six of these were known from earlier work. The total size of the proteins which appear to derive from the mer genes exceeds by a factor of 1.5, the coding capacity of this region without overlapping genes. Some, or all of these extra proteins may be chromosomal in origin, possibly derepressed in response to mercury gene products.

Bacteriophage lambda↗

Cloning and expression of AatII restriction-modification system in Escherichia coli.

The genes encoding the AatII restriction endonuclease and methylase from Acetobacter aceti have been cloned and expressed in Escherichia coli. The nucleotide sequences of aatIIM and aatIIR genes were determined. The aatIIM and aatIIR genes are 996 bp and 1038 bp, respectively, encoding the 331-aa methylase with a predicted molecular mass of 36.9 kDa, and the 345-aa AatII restriction endonuclease with a predicted molecular mass of 38.9 kDa. The two genes overlap by 4 base pairs and are transcribed in the same orientation. The aatIIRM genes are located next to a putative gene for plasmid mobilization. A stable overproducing strain was constructed, in which the aatIIM gene was expressed from a pSC101-derived plasmid. The aatIIR gene was inserted into a modified T7 expression vector that carries transcription terminators upstream from the T7 promoter. The recombinant AatII restriction endonuclease was purified to near homogeneity by chromatography through DEAE Sepharose, Heparin Sepharose, and phosphocellulose columns.

Acetobacter↗