Václav Pačes

Similar integration but different stability of Alus and LINEs in the human genome

Alus and LINEs (LINE1) are widespread classes of repeats that are very unevenly distributed in the human genome. The majority of GC-poor LINEs reside in the GC-poor isochores whereas GC-rich Alus are mostly present in GC-rich isochores. The discovery that LINES and Alus share similar target site duplication and a common AT-rich insertion site specificity raised the question as to why these two families of repeats show such a different distribution in the genome. This problem was investigated here by studying the isochore distributions of subfamilies of LINES and Alus characterized by different degrees of divergence from the consensus sequences, and of Alus, LINEs and pseudogenes located on chromosomes 21 and 22. Young Alus are more frequent in the GC-poor part of the genome than old Alus. This suggests that the gradual accumulation of Alus in GC-rich isochores has occurred because of their higher stability in compositionally matching chromosomal regions. Densities of Alus and LINEs increase and decrease, respectively, with increasing GC levels, except for the telomeric regions of the analyzed chromosomes. In addition to LINEs, processed pseudogenes are also more frequent in GC-poor isochores. Finally, the present results on Alu and LINE stability/exclusion predict significant losses of Alu DNA from the GC-poor isochores during evolution, a phenomenon apparently due to negative selection against sequences that differ from the isochore composition.

Assembly of the cnidarian camera-type eye from vertebrate-like components

Animal eyes are morphologically diverse. Their assembly, however, always relies on the same basic principle, i.e., photoreceptors located in the vicinity of dark shielding pigment. Cnidaria as the likely sister group to the Bilateria are the earliest branching phylum with a well developed visual system. Here, we show that camera-type eyes of the cubozoan jellyfish, Tripedalia cystophora, use genetic building blocks typical of vertebrate eyes, namely, a ciliary phototransduction cascade and melanogenic pathway. Our findings indicative of parallelism provide an insight into eye evolution. Combined, the available data favor the possibility that vertebrate and cubozoan eyes arose by independent recruitment of orthologous genes during evolution.

Biphenyl-Metabolizing Bacteria in the Rhizosphere of Horseradish and Bulk Soil Contaminated by Polychlorinated Biphenyls as Revealed by Stable Isotope Probing

ABSTRACT DNA-based stable isotope probing in combination with terminal restriction fragment length polymorphism was used in order to identify members of the microbial community that metabolize biphenyl in the rhizosphere of horseradish (Armoracia rusticana) cultivated in soil contaminated with polychlorinated biphenyls (PCBs) compared to members of the microbial community in initial, uncultivated bulk soil. On the basis of early and recurrent detection of their 16S rRNA genes in clone libraries constructed from [13C]DNA, Hydrogenophaga spp. appeared to dominate biphenyl catabolism in the horseradish rhizosphere soil, whereas Paenibacillus spp. were the predominant biphenyl-utilizing bacteria in the initial bulk soil. Other bacteria found to derive carbon from biphenyl in this nutrient-amended microcosm-based study belonged mostly to the class Betaproteobacteria and were identified as Achromobacter spp., Variovorax spp., Methylovorus spp., or Methylophilus spp. Some bacteria that were unclassified at the genus level were also detected, and these bacteria may be members of undescribed genera. The deduced amino acid sequences of the biphenyl dioxygenase α subunits (BphA) from bacteria that incorporated [13C]into DNA in 3-day incubations of the soils with [13C]biphenyl are almost identical to that of Pseudomonas alcaligenes B-357. This suggests that the spectrum of the PCB congeners that can be degraded by these enzymes may be similar to that of strain B-357. These results demonstrate that altering the soil environment can result in the participation of different bacteria in the metabolism of biphenyl.

Molecular cloning and expression of the human and mouse homologues of the Drosophila dachshund gene

Abstract Recent genetic analysis of the Drosophila dachshund (dac) gene has established that dac encodes a novel nuclear protein that is involved in both eye and leg development. In the Drosophila eye, dac expression appears to be controlled by the product of the eyeless/Pax6 gene. In order to analyze the Pax6 pathway in vertebrates we have isolated and characterized the cDNA and genomic clones corresponding to the human and mouse homologues of Drosophiladac. A full-length human cDNA encoding dachshund (DACH) encodes the 706 amino acids protein with predicted molecular weight of 73 kDa. A 109 amino acid domain located at the N-terminus of the DACH showed significant sequence and secondary structure homologies to the ski/sno oncogene products. Northern blot analysis found human DACH predominantly in adult kidney, heart, and placenta, with less expression detected in the brain, lung, skeletal muscle and pancreas. A panel of human cell lines was studied and most notably a large proportion of neuroblastomas expressed DACH mRNA. Mouse Dach encodes a protein of 751 amino acids with predicted molecular weight of 78 kDa that is 95% identical to the human DACH. RNase protection analysis showed the highest Dach mRNA expression in the adult mouse kidney and lung, whereas lower expression was detected in the brain and testis. RT/PCR analysis readily detected Dach mRNA in the adult mouse cornea and retina. Dach mRNA expression in the mouse E11.5 embryo was observed primarily in the fore and hind limbs, as well as in the somites.

Nucleotide sequence of the late region of Bacillus phage φ29 completes the 19285-bp sequence of φ29 genome. Comparison with the homologous sequence of phage PZA

The 12,177-bp nucleotide (nt) sequence of the late region of Bacillus phage luminal diameter 29 genome was determined. This sequence completes the entire 19,285-bp sequence of phage luminal diameter 29 DNA. Eleven open reading frames were found in this region, and these were assigned to eleven late genes. Ribosome-binding sites and a potential transcriptional promoter and terminator are considered. The nt sequence was compared to the homologous region of the closely related phage PZA and tolerated variations at the nt and amino acid (aa) level were evaluated. The most frequent changes are silent nt substitutions in the third position of codons, but aa substitutions are also found.

Pseudorabies virus immediate-early gene overlaps with an oppositely oriented open reading frame: Characterization of their promoter and enhancer regions

The immediate-early (IE) gene of pseudorabies virus (PRV) has recently been sequenced for two virus strains. To investigate IE gene regulation and to examine the genome segment reported to encode latency-related transcripts in opposite polarity to the IE gene, sequence analysis has been extended by 5 kb from each end of the IE gene. The IE promoter (P1) was found to be more complex than previously recognized: it consisted of nine imperfect repeats, each containing five to six different consensus elements for transcription factor binding. A second promoter (P2) was discovered downstream of the IE gene. It contained numerous octamer consensus sequences (ATGCAAAT) and recognition sites for transcription factor Sp1; specific binding of nuclear proteins to four Sp1 sites was detected. An open reading frame (ORF3) bordering on P2 was identified, oriented antiparallel to the IE gene. Potential enhancer elements (E3 and E4) were isolated by the enhancer trap technique. Linked to P1 and a CAT indicator gene, E3 acted as an enhancer and E4 as a silencer. The PRV IE gene product repressed transcription from its own promoter and activated the SV40 early promoter. The transactivating virion protein Vmw65 of HSV1 had an opposite effect on these promoters.

Complete genome sequence of the photosynthetic purple nonsulfur bacterium Rhodobacter capsulatus SB 1003.

Rhodobacter capsulatus SB 1003 belongs to the group of purple nonsulfur bacteria. Its genome consists of a 3.7-Mb chromosome and a 133-kb plasmid. The genome encodes genes for photosynthesis, nitrogen fixation, utilization of xenobiotic organic substrates, and synthesis of polyhydroxyalkanoates. These features made it a favorite research tool for studying these processes. Here we report its complete genome sequence.

HERVd: the Human Endogenous RetroViruses Database: update

An elaboration of HERVd (http://herv.img.cas.cz) is being carried out in two directions. One of them is the integration and better classification of families that diverge considerably from typical retroviral genomes. This leads to a more precise identification of members with individual families. The second improvement is better accessibility of the database and connection with human genome annotation.

M13 and pUC vectors with new unique restriction sites for cloning

Several vectors based on the widely used phage M13 and plasmid pUC were constructed. The vectors contain polylinkers (MCS) for DNA insertions with several new restriction sites (e.g., ApaI, NotI, StuI, SacII). Moreover, the NarI site in the nonessential part of the vector molecule was changed into a BssHII site, so that the NarI site in the MCS became unique.

Loss of the Response Regulator CtrA Causes Pleiotropic Effects on Gene Expression but Does Not Affect Growth Phase Regulation in Rhodobacter capsulatus

The purple nonsulfur photosynthetic bacterium Rhodobacter capsulatus has been extensively studied for its metabolic versatility as well as for production of a gene transfer agent called RcGTA. Production of RcGTA is highest in the stationary phase of growth and requires the response regulator protein CtrA. The CtrA protein in Caulobacter crescentus has been thoroughly studied for its role as an essential, master regulator of the cell cycle. Although the CtrA protein in R. capsulatus shares a high degree of sequence similarity with the C. crescentus protein, it is nonessential and clearly plays a different role in this bacterium. We have used transcriptomic and proteomic analyses of wild-type and ctrA mutant cultures to identify the genes dysregulated by the loss of CtrA in R. capsulatus. We have also characterized gene expression differences between the logarithmic and stationary phases of growth. Loss of CtrA has pleiotropic effects, with dysregulation of expression of approximately 6% of genes in the R. capsulatus genome. This includes all flagellar motility genes and a number of other putative regulatory proteins but does not appear to include any genes involved in the cell cycle. Quantitative proteomic data supported 88% of the CtrA transcriptome results. Phylogenetic analysis of CtrA sequences supports the hypothesis of an ancestral ctrA gene within the alphaproteobacteria, with subsequent diversification of function in the major alphaproteobacterial lineages.