A complex of the Srb8, -9, -10, and -11 transcriptional regulatory proteins from yeast

The Srb8, -9, -10, and -11 proteins of yeast have been isolated as a discrete, stoichiometric complex. The isolated complex phosphorylates the C-terminal domain (CTD) of the largest subunit of RNA polymerase II at serines 2 and 5. In addition to the previously reported human homologs of Srb10 and 11, we have identified TRAP230/ARC240 and TRAP240/ARC250 as the human homologs of Srb8 and Srb9, showing the entire Srb8/9/10/11 complex is conserved from yeast to humans.     

Genome-wide survey and expression profiling of CCCH-zinc finger family reveals a functional module in macrophage activation

Previously, we have identified a novel CCCH zinc finger protein family as negative regulators of macrophage activation. To gain an overall insight into the entire CCCH zinc finger gene family and to evaluate their potential role in macrophage activation, here we performed a genome-wide survey of CCCH zinc finger genes in mouse and human. Totally 58 CCCH zinc finger genes in mouse and 55 in human were identified and most of them have not been reported previously. Phylogenetic analysis revealed that the mouse CCCH family was divided into 6 groups. Meanwhile, we employed quantitative real-time PCR to profile their tissue expression patterns in adult mice. Clustering analysis showed that most of CCCH genes were broadly expressed in all of tissues examined with various levels. Interestingly, several CCCH genes Mbnl3, Zfp36l2, Zfp36, Zc3h12a, Zc3h12d, Zc3h7a and Leng9 were enriched in macrophage-related organs such as thymus, spleen, lung, intestine and adipose. Consistently, a comprehensive assessment of changes in expression of the 58 members of the mouse CCCH family during macrophage activation also revealed that these CCCH zinc finger genes were associated with the activation of bone marrow-derived macrophages by lipopolysaccharide. Taken together, this study not only identified a functional module of CCCH zinc finger genes in the regulation of macrophage activation but also provided the framework for future studies to dissect the function of this emerging gene family.     

Stress and developmental regulation of the yeast C-type cyclin Ume3p (Srb11p/Ssn8p).

The ume3-1 allele was identified as a mutation that allowed the aberrant expression of several meiotic genes (e.g. SPO11, SPO13) during mitotic cell division in Saccharomyces cerevisiae. Here we report that UME3 is also required for the full repression of the HSP70 family member SSA1. UME3 encodes a non-essential C-type cyclin (Ume3p) whose levels do not vary through the mitotic cell cycle. However, Ume3p is destroyed during meiosis or when cultures are subjected to heat shock. Ume3p mutants resistant to degradation resulted in a 2-fold reduction in SPO13 mRNA levels during meiosis, indicating that the down-regulation of this cyclin is important for normal meiotic gene expression. Mutational analysis identified two regions (PEST-rich and RXXL) that mediate Ume3p degradation. A third destruction signal lies within the highly conserved cyclin box, a region that mediates cyclin-cyclin-dependent kinase (Cdk) interactions. However, the Cdk activated by Ume3p (Ume5p) is not required for the rapid destruction of this cyclin. Finally, Ume3p destruction was not affected in mutants defective for ubiquitin-dependent proteolysis. These results support a model in which Ume3p, when exposed to heat shock or sporulation conditions, is targeted for destruction to allow the expression of genes necessary for the cell to respond correctly to these environmental cues.     

Genetic profile of the arylamine N-acetyltransferase 2 coding gene among individuals from two different regions of Brazil

Arylamine N-acetyltranferase 2 is the main enzyme responsible for the isoniazid metabolization into hepatotoxic intermediates and the degree of hepatotoxicity severity has been attributed to genetic variability in the NAT2 gene. The main goal of this study was to describe the genetic profile of the NAT2 gene in individuals from two different regions of Brazil: Rio de Janeiro and Goiás States. Therefore, after preparation of DNA samples from 404 individuals, genotyping of the coding region of NAT2 was performed by direct PCR sequencing. Thirteen previously described SNPs were detected in these Brazilian populations, from which seven: 191 G>A; 282 C>T; 341 T>C; 481 C>T; 590 G>A; 803 A>G and 857 G>A are the most frequent in other populations. The presence of so-called ethnic-specific SNPs in our population is in accordance with the Brazilians' multiple ancestry. Upon allele and genotype analysis, the most frequent NAT2 alleles were respectively NAT2*5B (33%), NAT2*6A (26%) and NAT2*4 (20%) being NAT2*5/*5 the more prevalent genotype (31.7%). These results clearly demonstrate the predominance in the studied Brazilian groups of NAT2 alleles associated with slow over the fast and intermediate acetylator genotypes. Additionally, in Rio de Janeiro, a significantly higher frequency of intermediate acetylation status was found when compared to Goiás (42.5% versus 25%) (p=0.05), demonstrating that different regions of a country with a population characterized by a multi-ethnic ancestry may present a large degree of variability in NAT2 allelic frequencies. This finding has implications in the determination of nationwide policies for use of appropriate anti-TB drugs.     

Structure of DNA polymerase beta with the mutagenic DNA lesion 8-oxodeoxyguanine reveals structural insights into its coding potential

Oxidative damage to DNA generates 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG). During DNA replication and repair synthesis, 8-oxodG can pair with cytosine or adenine. The ability to accurately replicate through this lesion depends on the DNA polymerase. We report the first structure of a polymerase with a promutagenic DNA lesion, 8-oxodG, in the confines of its active site. The modified guanine residue is in an anti conformation and forms Watson-Crick hydrogen bonds with an incoming dCTP. To accommodate the oxygen at C8, the 5'-phosphate backbone of the templating nucleotide flips 180 degrees. Thus, the flexibility of the template sugar-phosphate backbone near the polymerase active site is one parameter that influences the anti-syn equilibrium of 8-oxodG. Our results provide insights into the mechanisms employed by polymerases to select the complementary dNTP.     

Characterization of Hoxa-10/Hoxa-11 transheterozygotes reveals functional redundancy and regulatory interactions

Hox genes show related sequences and overlapping expression domains that often reflect functional redundancy as well as a common evolutionary origin. To accurately define their functions, it has become necessary to compare phenotypes of mice with single and multiple Hox gene mutations. Here, we focus on two Abd-B-type genes, Hoxa-10 and Hoxa-11, which are coexpressed in developing vertebrae, limbs, and reproductive tracts. To assess possible functional redundancy between these two genes, Hoxa-10/Hoxa-11 transheterozygotes were produced by genetic intercrosses and analyzed. This compound mutation resulted in synergistic defects in transheterozygous limbs and reproductive tracts, but not in vertebrae. In the forelimb, distal radial/ulnar thickening and pisiform/triangular carpal fusion were observed in 35 and 21% of transheterozygotes, respectively, but were effectively absent in Hoxa-10 and Hoxa-11 +/- forelimbs. In the hindlimb, distal tibial/fibular thickening and loss of tibial/fibular fusion were observed in >80% of transheterozygotes but in no Hoxa-10 or Hoxa-11 +/- hindlimbs, and all transheterozygotes displayed reduced medial patellar sesamoids, compared to modest incidences in Hoxa-10 and Hoxa-11 +/- mutants. Furthermore, while the reproductive tracts of Hoxa-10 and Hoxa-11 single heterozygous mutants of both sexes were primarily unaffected, male transheterozygotes displayed cryptorchidism and abnormal tortuosity of the ductus deferens, and female transheterozygotes exhibited abnormal uterotubal junctions and narrowing of the uterus. In addition we observed that the targeted mutations of Hoxa-10 and Hoxa-11 each affected the expression of the other gene in the developing prevertebra and reproductive tracts. These results provide a measure of the functional redundancy of Hoxa-10 and Hoxa-11 and a deeper understanding of the phenotypes resulting in the single mutants and help elucidate the regulatory interactions between these two genes.