Conserved regulatory elements of the promoter sequence of the gene rpoH of enteric bacteria

The rpoH regulatory region of different members of the enteric bacteria family was sequenced or downloaded from GenBank and compared. In addition, the transcriptional start sites of rpoH of Yersinia frederiksenii and Proteus mirabilis, two distant members of this family, were determined. Sequences similar to the σ70 promoters P1, P4 and P5, to the σE promoter P3 and to boxes DnaA1, DnaA2, cAMP receptor protein (CRP) boxes CRP1, CRP2 and box CytR present in Escherichia coli K12, were identified in sequences of closely related bacteria such as: E.coli, Shigella flexneri, Salmonella enterica serovar Typhimurium, Citrobacter freundii, Enterobacter cloacae and Klebsiella pneumoniae. In more distant bacteria, Y.frederiksenii and P.mirabilis, the rpoH regulatory region has a distal P1-like σ70 promoter and two proximal promoters: a heat-induced σE-like promoter and a σ70 promoter. Sequences similar to the regulatory boxes were not identified in these bacteria. This study suggests that the general pattern of transcription of the rpoH gene in enteric bacteria includes a distal σ70 promoter, >200 nt upstream of the initiation codon, and two proximal promoters: a heat-induced σE-like promoter and a σ70 promoter. A second proximal σ70 promoter under catabolite-regulation is probably present only in bacteria closely related to E.coli.

Human testis expresses a specific poly(A)-binding protein

In testis mRNA stability and translation initiation are extensively under the control of poly(A)-binding proteins (PABP). Here we have cloned a new human testis-specific PABP (PABP3) of 631 amino acids (70.1 kDa) with 92.5% identical residues to the ubiquitous PABP1. A northern blot of multiple human tissues hybridised with PABP3- and PABP1-specific oligonucleotide probes revealed two PABP3 mRNAs (2.1 and 2.5 kb) detected only in testis, whereas PABP1 mRNA (3.2 kb) was present in all tested tissues. In human adult testis, PABP3 mRNA expression was restricted to round spermatids, whereas PABP1 was expressed in these cells as well as in pachytene spermatocytes. PABP3-specific antibodies identified a protein of 70 kDa in human testis extracts. This protein binds poly(A) with a slightly lower affinity as compared to PABP1. The human PABP3 gene is intronless with a transcription start site 61 nt upstream from the initiation codon. A sequence of 256 bp upstream from the transcription start site drives the promoter activity of PABP3 and its tissue-specific expression. The expression of PABP3 might be a way to bypass PABP1 translational repression and to produce the amount of PABP needed for active mRNA translation in spermatids.

Complete structure of the human alpha-albumin gene, a new member of the serum albumin multigene family.

The nucleotide sequence of the human alpha-albumin gene, including 887 bp of the 5'-flanking region and 1311 bp of the 3-flanking region (24,454 in total), was determined from three overlapping lambda phage clones. The sequence spans 22,256 bp from the cap site to the polyadenylylation site, revealing a gene structure of 15 exons separated by 14 introns. The methionine initiation codon ATG is within exon 1; the termination codon TGA is within exon 14. Exon 15 is entirely untranslated and contains the polyadenylylation signal AATAAA. The deduced polypeptide chain is composed of a 21-amino-acid leader peptide, followed by 578 amino acids of the mature protein. There are seven repetitive DNA elements (Alu and Kpn) in the introns and 3-flanking region. The sizes of the 15 alpha-albumin exons match closely those of the albumin, alpha-fetoprotein, and vitamin D-binding protein genes. The exons are symmetrically placed within the three domains of the individual proteins, and they share a characteristic codon splitting pattern that is conserved among members of the gene family. The results provide strong evidence that alpha-albumin belongs to, and most likely completes with, the serum albumin gene family. Based on structural similarity, alpha-albumin appears to be most closely related to alpha-fetoprotein. The complete structure of this family of four tandemly linked genes provides a well-characterized approximately 200 kb locus in the 4q subcentromeric region of the human genome.

Foamy virus reverse transcriptase is expressed independently from the Gag protein.

In the foamy virus (FV) subgroup of retroviruses the pol genes are located in the +1 reading frame relative to the gag genes and possess potential ATG initiation codons in their 5' regions. This genome organization suggests either a + 1 ribosomal frameshift to generate a Gag-Pol fusion protein, similar to all other retroviruses studied so far, or new initiation of Pol translation, as used by pararetroviruses, to express the Pol protein. By using a genetic approach we have ruled out the former possibility and provide evidence for the latter. Two down-mutations (M53 and M54) of the pol ATG codon were found to abolish replication and Pol protein expression of the human FV isolate. The introduction of a new ATG in mutation M55, 3' to the down-mutated ATG of mutation M53, restored replication competence, indicating that the pol ATG functions as a translational initiation codon. Two nonsense mutants (M56 and M57), which functionally separated gag and pol with respect to potential frame-shifting sites, were also replication-competent, providing further genetic evidence that FVs express the Pol protein independently from Gag. Our results show that during a particular step of the replication cycle, FVs differ fundamentally from all other retroviruses.

A genetic screen identifies cellular factors involved in retroviral -1 frameshifting.

To identify cellular factors that function in -1 ribosomal frameshifting, we have developed assays in the yeast Saccharomyces cerevisiae to screen for host mutants in which frameshifting is specifically affected. Expression vectors have been constructed in which the mouse mammary tumor virus gag-pro frameshift region is placed upstream of the lacZ gene or the CUP1 gene so that the reporters are in the -1 frame relative to the initiation codon. These vectors have been used to demonstrate that -1 frameshifting is recapitulated in yeast in response to retroviral mRNA signals. Using these reporters, we have isolated spontaneous host mutants in two complementation groups, ifs1 and ifs2, in which frameshifting is increased 2-fold. These mutants are also hypersensitive to antibiotics that target the 40S ribosomal subunit. We have cloned the IFS1 gene and shown that it encodes a previously undescribed protein of 1091 aa with clusters of acidic residues in the carboxyl-terminal region. Haploid cells lacking 82% of the IFS1 open reading frame are viable and phenotypically identical to ifs1-1 mutants. This approach could help identify potential targets for antiretroviral agents.

Next-generation sequencing of iron-metabolism related genes in Portuguese patients with iron overload: novel pathogenic genetic variants

Objective: In Southern European countries up to one-third of the patients with hereditary hemochromatosis (HH) do not present the common HFE risk genotype. In order to investigate the molecular basis of these cases we have designed a gene panel for rapid and simultaneous analysis of 6 HH-related genes (HFE, TFR2, HJV, HAMP, SLC40A1 and FTL) by next-generation sequencing (NGS). Materials and Methods: Eighty-eight iron overload Portuguese patients, negative for the common HFE mutations, were analysed. A TruSeq Custom Amplicon kit (TSCA, by Illumina) was designed in order to generate 97 amplicons covering exons, intron/exon junctions and UTRs of the mentioned genes with a cumulative target sequence of 12115bp. Amplicons were sequenced in the MiSeq instrument (IIlumina) using 250bp paired-end reads. Sequences were aligned against human genome reference hg19 using alignment and variant caller algorithms in the MiSeq reporter software. Novel variants were validated by Sanger sequencing and their pathogenic significance were assessed by in silico studies. Results: We found a total of 55 different genetic variants. These include novel pathogenic missense and splicing variants (in HFE and TFR2), a very rare variant in IRE of FTL, a variant that originates a novel translation initiation codon in the HAMP gene, among others. Conclusion: The merging of TSCA methodology and NGS technology appears to be an appropriate tool for simultaneous and fast analysis of HH-related genes in a large number of samples. However, establishing the clinical relevance of NGS-detected variants for HH development remains a hard-working task, requiring further functional studies.

Differential effects on the hepatitis C virus (HCV) internal ribosome entry site by vitamin B-12 and the HCV core protein

To investigate the role of the hepatitis C virus internal ribosome entry site (HCV IRES) domain IV in translation initiation and regulation, two chimeric IRES elements were constructed to contain the reciprocal domain IV in the otherwise HCV and classical swine fever virus IRES elements. This permitted an examination of the role of domain IV in the control of HCV translation. A specific inhibitor of the HCV IRES, vitamin B-12 was shown to inhibit translation directed by all IRES elements which contained domain IV from the HCV and the GB virus B IRES elements, whereas the HCV core protein could only suppress translation from the wild-type HCV IRES. Thus, the mechanisms of translation inhibition by vitamin B-12 and the core protein differ, and they target different regions of the IRES.

Incorporating a TEV cleavage site reduces the solubility of nine recombinant mouse proteins

Failure to express soluble proteins in bacteria is mainly attributed to the properties of the target protein itself, as well as the choice of the vector, the purification tag and the linker between the tag and protein, and codon usage. The expression of proteins with fusion tags to facilitate subsequent purification steps is a widely used procedure in the production of recombinant proteins. However, the additional residues can affect the properties of the protein; therefore, it is often desirable to remove the tag after purification. This is usually done by engineering a cleavage site between the tag and the encoded protein that is recognised by a site-specific protease, such as the one from tobacco etch virus (TEV). In this study, we investigated the effect of four different tags on the bacterial expression and solubility of nine mouse proteins. Two of the four engineered constructs contained hexahistidine tags with either a long or short linker. The other two constructs contained a TEV cleavage site engineered into the linker region. Our data show that inclusion of the TEV recognition site directly downstream of the recombination site of the Invitrogen Gateway vector resulted, in a loss of solubility of the nine mouse proteins. Our work suggests that one needs to be very careful when making modifications to expression vectors and combining different affinity and fusion tags and cleavage sites: (c) 2006 Elsevier Inc. All rights reserved.

A nonantisense sequence-selective effect of a phosphorothioate oligodeoxynucleotide directed against the epidermal growth factor receptor in A431 cells

The overexpression of epidermal growth factor receptor (EGFr) has been implicated as a causative factor and a poor prognostic marker in a number of carcinomas. Therefore, strategies that down-regulate EGFr expression may be therapeutically useful. We designed antisense ODNs complementary to the initiation codon region of the EGFr mRNA and evaluated their efficacy in several tumor-derived cells, including the A431 cell line that express amplified levels of EGFr. A 15-mer phosphorothioate (PS) antisense ODN (erbB1AS15) induced a concentration-dependent reduction in proliferation that was accompanied by a change in the morphology of A431 cells into more tightly clustered and discrete colonies. A 15-mer sense (PS) control oligodeoxynucleotide (ODN) and a phosphodiester (PO) version of erbB1AS15 had little or no effect on cell number of morphology, and erbB1AS15 (PS) did not induce these effects in control cell lines expressing lower levels of EGFr. The effects of erbB1AS15 (PS) on A431 cells were not mediated by a true antisense mechanism in that there was no reduction in the level of EGFr mRNA or protein over a 24-hr period, as determined by Northern and Western blotting, respectively. However, autophosphorylation of the receptor was significantly reduced by erbB1AS15 (PS) and not by control ODNs. The results of further studies suggested that this effect was mediated by a direct, dose-dependent inhibition of the EGFr tyrosine kinase enzyme and was not due to impairment of either ligand-binding or receptor dimerization. These data suggest that erbB1AS15 (PS) can inhibit proliferation and alter the morphology of A431 cells by a sequence-selective, but nonantisense mechanism affecting receptor tyrosine kinase activity.

Study of sunless tanning formulas using molted snake skin as an alternative membrane model

Sunless tanning formulas have become increasingly popular in recent years for their ability to give people convincing tans without the dangers of skin cancer. Most sunless tanners currently on the market contain dihydroxyacetone (DHA), a keto sugar with three carbons. The temporary pigment provided by these formulasis designed to resemble a UV-induced tan. This study evaluated the effectiveness of carbomer gels and cold process self emulsifying bases on skin pigmentation, using different concentrations of a chemical system composed of DHA and N-acetyl tyrosine, which are found in moulted snake skins and their effectiveness was tested by Mexameter (R) MX 18. Eight different sunless tanning formulas were developed, four of which were gels and four of which were emulsions (base, base plus 4.0%, 5.0% and 6.0% (w/w) of a system of DHA and N-acetyl tyrosine). Tests to determine the extent of artificial tanning were done by applying 30 mg cm(-2) of each formula onto standard sizes of moulted snake skin (2.0 cm x 3.0 cm). A Mexameter (R) MX 18 was used to evaluate the extent of coloration in the moulted snake skin at T(0) (before the application) and after 24, 48, 72, 168, 192 and 216 h. The moulted snake skins can be used as an alternative membrane model for in vitro sunless tanning efficacy tests due to their similarity to the human stratum corneum. The DHA concentration was found to influence the initiation of the pigmentation in both sunless tanning systems (emulsion and gel) as well as the time required to increases by a given amount on the tanning index. In the emulsion system, the DHA concentration also influenced the final value on the tanning index. The type of system (emulsion or gel) has no influence on the final value in the tanning index after 216 h for samples with the same DHA concentration.

Genetic analysis of complement C1s deficiency associated with systemic lupus erythernatosus highlights alternative splicing of normal C1s gene

Deficiencies of complement proteins of the classical pathway are strongly associated with the development of autoimmune diseases. Deficiency of Clr has been observed to occur concomitantly with deficiency in Cls and 9 out of 15 reported cases presented systemic lupus erythernatosus (SLE). Here, we describe a family in which all four children are deficient in Cls but only two of them developed SLE. Hemolytic activity mediated by the alternative and the lectin pathways were normal, but classical pathway activation was absent in all children`s sera. Cls was undetectable, while in the parents` sera it was lower than in the normal controls. The levels of Clr observed in the siblings and parents sera were lower than in the control, while the concentrations of other complement proteins (C3, C4, MBL and MASP-2) were normal in all family members. Impairment of Cls synthesis was observed in the patients` fibroblasts when analyzed by confocal microscopy. We show that all four siblings are homozygous for a mutation at position 938 in exon 6 of the Cls cDNA that creates a premature stop codon. Our investigations led us to reveal the presence of previously uncharacterized splice variants of Cls mRNA transcripts in normal human cells. These variants are derived from the skipping of exon 3 and from the use of an alternative 3` splice site within intron I which increases the size of exon 2 by 87 nucleotides. (c) 2007 Elsevier Ltd. All rights reserved.

Predicting cave initiation and propagation

The most widely used method for predicting the onset of continuous caving is Laubscher's caving chart. A detailed examination of this method was undertaken which concluded that it had limitations which may impact on results, particularly when dealing with stronger rock masses that are outside current experience. These limitations relate to inadequate guidelines for adjustment factors to rock mass rating (RMR), concerns about the position on the chart of critical case history data, undocumented changes to the method and an inadequate number of data points to be confident of stability boundaries. A review was undertaken on the application and reliability of a numerical method of assessing cavability. The review highlighted a number of issues, which at this stage, make numerical continuum methods problematic for predicting cavability. This is in particular reference to sensitivity to input parameters that are difficult to determine accurately and mesh dependency. An extended version of the Mathews method for open stope design was developed as an alternative method of predicting the onset of continuous caving. A number of caving case histories were collected and analyzed and a caving boundary delineated statistically on the Mathews stability graph. The definition of the caving boundary was aided by the existence of a large and wide-ranging stability database from non-caving mines. A caving rate model was extrapolated from the extended Mathews stability graph but could only be partially validated due to a lack of reliable data.

Improved efficiency of a Salmonella-based vaccine against human papillomavirus type 16 virus-like particles achieved by using a codon-optimized version of L1.

Cervical cancer results from cervical infection by human papillomaviruses (HPVs), especially HPV16. An effective vaccine against these HPVs is expected to have a dramatic impact on the incidence of this cancer and its precursor lesions. The leading candidate, a subunit prophylactic HPV virus-like particle (VLP) vaccine, can protect women from HPV infection. An alternative improved vaccine that avoids parenteral injection, that is efficient with a single dose, and that induces mucosal immunity might greatly facilitate vaccine implementation in different settings. In this study, we have constructed a new generation of recombinant Salmonella organisms that assemble HPV16 VLPs and induce high titers of neutralizing antibodies in mice after a single nasal or oral immunization with live bacteria. This was achieved through the expression of a HPV16 L1 capsid gene whose codon usage was optimized to fit with the most frequently used codons in Salmonella. Interestingly, the high immunogenicity of the new recombinant bacteria did not correlate with an increased expression of L1 VLPs but with a greater stability of the L1-expressing plasmid in vitro and in vivo in absence of antibiotic selection. Anti-HPV16 humoral and neutralizing responses were also observed with different Salmonella enterica serovar Typhimurium strains whose attenuating deletions have already been shown to be safe after oral vaccination of humans. Thus, our findings are a promising improvement toward a vaccine strain that could be tested in human volunteers.

Developmental stage-specific expression of cyclic adenosine 3',5'-monophosphate response element-binding protein CREB during spermatogenesis involves alternative exon splicing.

Spermatogenesis is a temporally regulated developmental process by which the gonadotropin-responsive somatic Sertoli and Leydig cells act interdependently to direct the maturation of the germinal cells. The metabolism of Sertoli and Leydig cells is regulated by the pituitary gonadotropins FSH and LH, which, in turn, activate adenylate cyclase. Because the cAMP-second messenger pathway is activated by FSH and LH, we postulated that the cAMP-responsive element-binding protein (CREB) plays a physiological role in Sertoli and Leydig cells, respectively. Immunocytochemical analyses of rat testicular sections show a remarkably high expression of CREB in the haploid round spermatids and, to some extent, in pachytene spermatocytes and Sertoli cells. Although most of the CREB antigen is detected in the nuclei, some CREB antigen is also present in the cytoplasm. Remarkably, the cytoplasmic CREB results from the translation of a unique alternatively spliced transcript of the CREB gene that incorporates an exon containing multiple stop codons inserted immediately up-stream of the exons encoding the DNA-binding domain of CREB. Thus, the RNA containing the alternatively spliced exon encodes a truncated transcriptional transactivator protein lacking both the DNA-binding domain and nuclear translocation signal of CREB. Most of the CREB transcripts detected in the germinal cells contain the alternatively spliced exon, suggesting a function of the exon to modulate the synthesis of CREB. In the Sertoli cells we observed a striking cyclical (12-day periodicity) increase in the levels of CREB mRNA that coincides with the splicing out of the restrictive exon containing the stop codons. Because earlier studies established that FSH-stimulated cAMP levels in Sertoli cells are also cyclical, and the CREB gene promoter contains cAMP-responsive enhancers, we suggest that the alternative RNA splicing controls a positive autoregulation of CREB gene expression mediated by cAMP.

Fine-Tuning Translation Kinetics Selection as the Driving Force of Codon Usage Bias in the Hepatitis A Virus Capsid

Hepatitis A virus (HAV), the prototype of genus Hepatovirus, has several unique biological characteristics that distinguish it from other members of the Picornaviridae family. Among these, the need for an intact eIF4G factor for the initiation of translation results in an inability to shut down host protein synthesis by a mechanism similar to that of other picornaviruses. Consequently, HAV must inefficiently compete for the cellular translational machinery and this may explain its poor growth in cell culture. In this context of virus/cell competition, HAV has strategically adopted a naturally highly deoptimized codon usage with respect to that of its cellular host. With the aim to optimize its codon usage the virus was adapted to propagate in cells with impaired protein synthesis, in order to make tRNA pools more available for the virus. A significant loss of fitness was the immediate response to the adaptation process that was, however, later on recovered and more associated to a re-deoptimization rather than to an optimization of the codon usage specifically in the capsid coding region. These results exclude translation selection and instead suggest fine-tuning translation kinetics selection as the underlying mechanism of the codon usage bias in this specific genome region. Additionally, the results provide clear evidence of the Red Queen dynamics of evolution since the virus has very much evolved to re-adapt its codon usage to the environmental cellular changing conditions in order to recover the original fitness.