Identification of the minimal promoter region of the mouse NKX5-3, a transcription factor implicated in eye development.

Early ocular development is controlled by a complex network of transcription factors, cell cycle regulators, and diffusible signalling molecules. Together, these molecules regulate cell proliferation and apoptosis, and specify retinal fate. NKX5-3 is a homeobox transcription factor implicated in eye development. The analysis of the 5'-flanking region of the mouse Nkx5-3 gene revealed a predicted TATA-less promoter sequence between -416 and -166 of the translation start site. To functionally characterise Nkx5-3 promoter activity, serial deletions of the promoter sequence were introduced in pGL-3 basic vector and promoter activity of these 5'- and 3'-deleted constructions was tested in HeLa and CHO cells. Transactivation assays identified a region between -350 and -296 exhibiting promoter-like activity. Combined analysis by deletions and point mutations showed that this sequence, containing multiple Sp1 binding sites was necessary to promote transcriptional activity. Binding of Sp1 to this region was confirmed by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation, using an antibody specific for Sp1. Altogether, these results demonstrated that the immediate upstream region of Nkx5-3 gene possessed a strong intrinsic promoter activity in vitro, suggesting a potential role in Nkx5-3 transcription in vivo.

Efficient targeted transcript discovery via array-based normalization of RACE libraries.

Rapid amplification of cDNA ends (RACE) is a widely used approach for transcript identification. Random clone selection from the RACE mixture, however, is an ineffective sampling strategy if the dynamic range of transcript abundances is large. To improve sampling efficiency of human transcripts, we hybridized the products of the RACE reaction onto tiling arrays and used the detected exons to delineate a series of reverse-transcriptase (RT)-PCRs, through which the original RACE transcript population was segregated into simpler transcript populations. We independently cloned the products and sequenced randomly selected clones. This approach, RACEarray, is superior to direct cloning and sequencing of RACE products because it specifically targets new transcripts and often results in overall normalization of transcript abundance. We show theoretically and experimentally that this strategy leads indeed to efficient sampling of new transcripts, and we investigated multiplexing the strategy by pooling RACE reactions from multiple interrogated loci before hybridization.

Effect of an 8-weeks aerobic training program in elderly on oxidative stress and HSP72 expression in leukocytes during antioxidant supplementation.

OBJECTIVE: To investigate the effect of aerobic training in the context of antioxidant supplementation on systemic oxidative stress and leukocytes heat shock protein (Hsp)72 expression in the elderly. DESIGN: Sixteen septuagenarians (8 males and 8 females, mean age 74.6) were supplemented with Vitamin C and E (respectively 500 and 100mg per day) and randomly assigned either to sedentary (AS) or individualized aerobically trained (AT) group for 8 weeks. METHODS: Plasma Vitamin C and E concentrations and aerobic fitness, as well as resting and post graded exercise (GXT) Hsp72 expression in leukocytes, plasma levels of thiobarbituric acid reactive substances (TBARS) and advanced oxidation protein product (AOPP) were measured pre and post training / supplementation. RESULTS: At the end of the intervention, the two groups showed a significant increase in resting plasma vitamin C and E (approximately 50 and 20% increase respectively) and a significant decrease in both resting and post GXT plasma TBARS and AOPP (approximately 25 and 20% decrease respectively). These changes were of similar magnitude in the two groups. The reduced oxidative stress was concomitant with a 15% decreased expression of Hsp72 in monocytes and granulocytes in both groups. CONCLUSION: This study provides evidence that in elderly, increased concentration of antioxidant vitamins C and E is associated with a reduction in oxidative stress and leukocytes Hsp72. In this context, 8 weeks of aerobic training has no impact on oxidative stress or leukocytes Hsp72 expression in elderly people.

Long sequence duplications, repeats, and palindromes in HIV-1 gp120: length variation in V4 as the product of misalignment mechanism.

We have shown that indels in gp120 V4 are associated to the presence of duplicated and palindromic sequences, suggesting that they may be produced by strand-slippage misalignment mechanism. Indels in V4 involved region-specific duplications 9 to 15 bp long, and repeats of various lengths, associated to trinucleotides AAT. No duplications were found in V3 and C3. The frequency of palindromic sequences in individual genes was found to be significantly higher in gp120 (p < or = 3.00E-7), and significantly lower in Tat (p < or = 9.00E-7) than the average frequency calculated over the full genome. The finding of elements of misalignment in association with indels in V4 suggests that these mutations may occur in proviral DNA after integration of HIV into the host genome. It also implies that occurrence of large indels in gp120 is not random but is directed by the presence and distribution of elements of misalignment in the HIV genome.

Nuclear translocation and DNA recognition signals colocalized within the bZIP domain of cyclic adenosine 3',5'-monophosphate response element-binding protein CREB.

CREB is a cAMP-responsive nuclear DNA-binding protein that binds to cAMP response elements and stimulates gene transcription upon activation of the cAMP signalling pathway. The protein consists of an amino-terminal transcriptional transactivation domain and a carboxyl-terminal DNA-binding domain (bZIP domain) comprised of a basic region and a leucine zipper involved in DNA recognition and dimerization, respectively. Recently, we discovered a testis-specific transcript of CREB that contains an alternatively spliced exon encoding multiple stop codons. CREB encoded by this transcript is a truncated protein lacking the bZIP domain. We postulated that the antigen detected by CREB antiserum in the cytoplasm of germinal cells is the truncated CREB that must also lack its nuclear translocation signal (NTS). To test this hypothesis we prepared multiple expression plasmids encoding carboxyl-terminal deletions of CREB and transiently expressed them in COS-1 cells. By Western immunoblot analysis as well as immunocytochemistry of transfected cells, we show that CREB proteins truncated to amino acid 286 or shorter are sequestered in the cytoplasm, whereas a CREB of 295 amino acids is translocated into the nucleus. Chimeric CREBs containing a heterologous NTS fused to the first 248 or 261 amino acids of CREB are able to drive the translocation of the protein into the nucleus. Thus, the nine amino acids in the basic region involved in DNA recognition between positions 287 and 295 (RRKKKEYVK) of CREB contain the NTS. Further, mutation of the lysine at position 290 in CREB to an asparagine diminishes nuclear translocation of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of cancer/testis-antigen genes by massively parallel signature sequencing.

Massively parallel signature sequencing (MPSS) generates millions of short sequence tags corresponding to transcripts from a single RNA preparation. Most MPSS tags can be unambiguously assigned to genes, thereby generating a comprehensive expression profile of the tissue of origin. From the comparison of MPSS data from 32 normal human tissues, we identified 1,056 genes that are predominantly expressed in the testis. Further evaluation by using MPSS tags from cancer cell lines and EST data from a wide variety of tumors identified 202 of these genes as candidates for encoding cancer/testis (CT) antigens. Of these genes, the expression in normal tissues was assessed by RT-PCR in a subset of 166 intron-containing genes, and those with confirmed testis-predominant expression were further evaluated for their expression in 21 cancer cell lines. Thus, 20 CT or CT-like genes were identified, with several exhibiting expression in five or more of the cancer cell lines examined. One of these genes is a member of a CT gene family that we designated as CT45. The CT45 family comprises six highly similar (>98% cDNA identity) genes that are clustered in tandem within a 125-kb region on Xq26.3. CT45 was found to be frequently expressed in both cancer cell lines and lung cancer specimens. Thus, MPSS analysis has resulted in a significant extension of our knowledge of CT antigens, leading to the discovery of a distinctive X-linked CT-antigen gene family.

Dynamics of multiple lin gene expression in Sphingomonas paucimobilis B90A in response to different hexachlorocyclohexane isomers.

Sphingomonas paucimobilis B90A is able to degrade the alpha-, beta-, gamma-, and delta-isomers of hexachlorocyclohexane (HCH). It contains the genes linA, linB, linC, linD, linE, and linR, which have been implicated in HCH degradation. In this study, dynamic expression of the lin genes was measured in chemostat-grown S. paucimobilis B90A by RNA dot blot hybridization and real-time reverse transcriptase PCR upon exposure to a pulse of different HCH isomers. Irrespective of the addition of HCH, linA, linB, and linC were all expressed constitutively. In contrast, linD and linE were induced with alpha-HCH (2 mg/liter) and gamma-HCH (7 mg/liter). A sharp increase in mRNA levels for linD and linE was observed from 10 to 45 min after the addition of alpha- or gamma-HCH. Induction of linD and linE was not detectable upon the addition of 0.7 mg of gamma-HCH per liter, although the compound was degraded by the cells. The addition of beta-HCH (5 mg/liter) or delta-HCH (20 mg/liter) did not lead to linE and linD induction, despite the fact that 50% of the compounds were degraded. This suggests that degradation of beta- and delta-HCH proceeds by a different pathway than that of alpha- and gamma-HCH.

Contamination of genomic databases by HIV-1 and its possible consequences. A study in Bioinformatics.

Peer-reviewed

Structure of the glycosyl-phosphatidylinositol membrane anchor of the Leishmania major promastigote surface protease.

In common with many other plasma membrane glycoproteins of eukaryotic origin, the promastigote surface protease (PSP) of the protozoan parasite Leishmania contains a glycosyl-phosphatidylinositol (GPI) membrane anchor. The GPI anchor of Leishmania major PSP was purified following proteolysis of the PSP and analyzed by two-dimensional 1H-1H NMR, compositional and methylation linkage analyses, chemical and enzymatic modifications, and amino acid sequencing. From these results, the structure of the GPI-containing peptide was found to be Asp-Gly-Gly-Asn-ethanolamine-PO4-6Man alpha 1-6Man alpha 1-4GlcN alpha 1-6myo-inositol-1-PO4-(1-alkyl-2-acyl-glycerol). The glycan structure is identical to the conserved glycan core regions of the GPI anchor of Trypanosoma brucei variant surface glycoprotein and rat brain Thy-1 antigen, supporting the notion that this portion of GPIs are highly conserved. The phosphatidylinositol moiety of the PSP anchor is unusual, containing a fully saturated, unbranched 1-O-alkyl chain (mainly C24:0) and a mixture of fully saturated unbranched 2-O-acyl chains (C12:0, C14:0, C16:0, and C18:0). This lipid composition differs significantly from those of the GPIs of T. brucei variant surface glycoprotein and mammalian erythrocyte acetylcholinesterase but is similar to that of a family of glycosylated phosphoinositides found uniquely in Leishmania.

RDM1, a novel RNA recognition motif (RRM)-containing protein involved in the cell response to cisplatin in vertebrates.

A variety of cellular proteins has the ability to recognize DNA lesions induced by the anti-cancer drug cisplatin, with diverse consequences on their repair and on the therapeutic effectiveness of this drug. We report a novel gene involved in the cell response to cisplatin in vertebrates. The RDM1 gene (for RAD52 Motif 1) was identified while searching databases for sequences showing similarities to RAD52, a protein involved in homologous recombination and DNA double-strand break repair. Ablation of RDM1 in the chicken B cell line DT40 led to a more than 3-fold increase in sensitivity to cisplatin. However, RDM1-/- cells were not hypersensitive to DNA damages caused by ionizing radiation, UV irradiation, or the alkylating agent methylmethane sulfonate. The RDM1 protein displays a nucleic acid binding domain of the RNA recognition motif (RRM) type. By using gel-shift assays and electron microscopy, we show that purified, recombinant chicken RDM1 protein interacts with single-stranded DNA as well as double-stranded DNA, on which it assembles filament-like structures. Notably, RDM1 recognizes DNA distortions induced by cisplatin-DNA adducts in vitro. Finally, human RDM1 transcripts are abundant in the testis, suggesting a possible role during spermatogenesis.

cis- and trans-acting elements involved in reactivation of vaccinia virus early transcription.

We have previously shown that transcription from the vaccinia virus 7.5K early promoter is reactivated late in infection (J. Garcés, K. Masternak, B. Kunz, and R. Wittek, J. Virol. 67:5394-5401, 1993). To identify the sequence elements mediating reactivation, we constructed recombinant viruses harboring deletions, substitutions, or insertions in the 7.5K promoter or its flanking regions. The analysis of these viruses showed that sequences both upstream as well as downstream of the transcription initiation site contribute to reactivation of the 7.5K promoter. We tested whether reactivation could be explained by a high affinity of vaccinia virus early transcription factor to reactivated promoters. Bandshift experiments using purified protein showed that promoters which bind the factor with high affinity in general also have high early transcriptional activity. However, no correlation was found between affinity of the factor and reactivation. Interestingly, overexpression of recombinant early transcription factor in vaccinia virus-infected cells resulted in a shutdown of late transcription and in reactivation of promoters, which are normally not reactivated.

Evidence of recombination in putative ancient asexuals.

Ancient asexuals have been considered to be a contradiction of the basic tenets of evolutionary theory. Barred from rearranging genetic variation by recombination, their reduced number of gene arrangements is thought to hamper their response to changing environments. For the same reason, it should be difficult for them to avoid the build-up of deleterious mutations. Several groups of taxonomically diverse organisms are thought to be ancient asexuals, although clear evidence for or against the existence of recombination events is scarce. Several methods have recently been developed for predicting recombination events by analyzing aligned sequences of a given region of DNA that all originate from one species. The methods are based on phylogenetic, substitution, and compatibility analyses. Here we present the results of analyses of sequence data from different loci studied in several groups of evolutionarily distant species that are considered to be ancient asexuals, using seven different types of analysis. The groups of organisms were the arbuscular mycorrhizal fungi (Glomales), Darwinula stevensoni (Darwinuloidea crustacean ostracods) and the bdelloid rotifers (Bdelloidea), which are thought to have been asexual for the last 400, 25-100, and 35-40 Myr, respectively. The seven different analytical methods evaluated the evolutionary relationships among haplotypes, and these methods had previously been shown to be reliable for predicting the occurrence of recombination events. Despite the different degree of genetic variation among the different groups of organisms, at least some evidence for recombination was found in all species groups. In particular, predictions of recombination events in the arbuscular mycorrhizal fungi were frequent. Predictions of recombination were also found for sequence data that have previously been used to infer the absence of recombination in bdelloid rotifers. Although our results have to be taken with some caution because they could signal very ancient recombination events or possibly other genetic variation of nonrecombinant origin, they suggest that some cryptic recombination events may exist in these organisms.

Analysis of parapoxvirus genomes.

Eight stomatitis papulosa (SP), four orf and two milker's nodes (MN) virus isolates were compared by restriction enzyme analysis. Considerable genetic heterogeneity was found not only between isolates belonging to the three different taxonomic groups but also between members of the same group. This heterogeneity precludes classification of parapoxviruses simply by comparison of their DNA cleavage patterns. Restriction maps were therefore prepared for 12 parapoxvirus DNAs. Fragments from defined regions of the genome were then selected and used as probes for cross-hybridizations to all other parapoxvirus DNAs. DNA fragments derived from an internal region of the genome hybridized strongly to all parapoxvirus isolates examined. In contrast, cross-hybridization of the end region of the DNA molecule was observed only between members of the same virus group. Molecular hybridization as a means of classifying parapoxvirus isolates is discussed.