Current Insights in Elucidation of Possible Molecular Mechanisms of the Juvenile Form of Batten Disease.

The neuronal ceroid lipofuscinoses (NCLs) collectively constitute one of the most common forms of inherited childhood-onset neurodegenerative disorders. They form a heterogeneous group of incurable lysosomal storage diseases that lead to blindness, motor deterioration, epilepsy, and dementia. Traditionally the NCL diseases were classified according to the age of disease onset (infantile, late-infantile, juvenile, and adult forms), with at least 13 different NCL varieties having been described at present.

The Human Isoform of RNA Polymerase II Subunit hRPB11bα Specifically Interacts with Transcription Factor ATF4.

Rpb11 subunit of RNA polymerase II of Eukaryotes is related to N-terminal domain of eubacterial α subunit and forms a complex with Rpb3 subunit analogous to prokaryotic α homodimer, which is involved in RNA polymerase assembly and promoter recognition. In humans, a gene family has been identified that potentially encodes several hRPB11 proteins differing mainly in their short C-terminal regions. The functions of the different human specific isoforms are still mainly unknown.

Molecular mechanisms of the juvenile form of Batten disease: important role of MAPK signaling pathways (ERK1/ERK2, JNK and p38) in pathogenesis of the malady.

Background: Mutations in the CLN3 gene lead to so far an incurable juvenile-onset neuronal ceroid lipofuscinosis (JNCL) or Batten disease that starts at the age of 4-6 years with a progressive retinopathy leading to blindness. Motor disturbances, epilepsy and dementia manifest during several following years. Most JNCL patients carry the same 1.

i-Clamp phenoxazine for the fine tuning of DNA i-motif stability.

Non-canonical DNA structures are widely used for regulation of gene expression, in DNA nanotechnology and for the development of new DNA-based sensors. I-motifs (iMs) are two intercalated parallel duplexes that are held together by hemiprotonated C-C base pairs. Previously, iMs were used as an accurate sensor for intracellular pH measurements.

A key enzyme of animal steroidogenesis can function in plants enhancing their immunity and accelerating the processes of growth and development.

Background: The initial stage of the biosynthesis of steroid hormones in animals occurs in the mitochondria of steroidogenic tissues, where cytochrome P450 (CYP11A1) encoded by the CYP11A1 gene catalyzes the conversion of cholesterol into pregnenolone - the general precursor of all the steroid hormones, starting with progesterone. This stage is missing in plants where mitochondrial cytochromes P450 (the mito CYP clan) have not been found. Generating transgenic plants with a mitochondrial type P450 from animals would offer an interesting option to verify whether plant mitochondria could serve as another site of P450 monooxygenase reaction for the steroid hormones biosynthesis.

Different roles of glutathione in copper and zinc chelation in Brassica napus roots.

We investigated the specific features of copper and zinc excess action on the roots of canola (Brassica napus L.) plants. Copper rapidly accumulated in canola root cells and reached saturation during several hours of treatment, whereas the root zinc content increased relatively slowly.

Synthesis of oligonucleotides containing novel G-clamp analogue with C8-tethered group in phenoxazine ring: Implication to qPCR detection of the low-copy Kemerovo virus dsRNA.

Nowadays modified oligonucleotides are widely used in diagnostics and as novel therapeutics. Introduction of modified or unnatural residues into oligonucleotides allows fine tuning of their binding properties to complementary nucleic acids and leads to improved stability both in vitro and in vivo. Previously it was demonstrated that insertion of phenoxazine nucleotides with various groups in C9-position into oligonucleotides leads to a significant increase of duplex stability with complementary DNA and RNA.

A minor isoform of the human RNA polymerase II subunit hRPB11 (POLR2J) interacts with several components of the translation initiation factor eIF3.

Using the yeast two-hybrid (YTH) system we have uncovered interaction of the hRPB11cα minor isoform of Homo sapiens RNA polymerase II hRPB11 (POLR2J) subunit with three different subunits of the human translation initiation factor eIF3 (hEIF3): eIF3a, eIF3i, and eIF3m. One variant of eIF3m identified in the study is the product of translation of alternatively spliced mRNA. We have named a novel isoform of this subunit eIF3mβ.

Ancient origin, functional conservation and fast evolution of DNA-dependent RNA polymerase III.

RNA polymerase III contains seventeen subunits in yeasts (Saccharomyces cerevisiae and Schizosaccharomyces pombe) and in human cells. Twelve of them are akin to the core RNA polymerase I or II. The five other are RNA polymerase III-specific and form the functionally distinct groups Rpc31-Rpc34-Rpc82 and Rpc37-Rpc53.

Distinct regions of RPB11 are required for heterodimerization with RPB3 in human and yeast RNA polymerase II.

In Saccharomyces cerevisiae, RNA polymerase II assembly is probably initiated by the formation of the RPB3-RPB11 heterodimer. RPB3 is encoded by a single copy gene in the yeast, mouse and human genomes. The RPB11 gene is also unique in yeast and mouse, but in humans a gene family has been identified that potentially encodes several RPB11 proteins differing mainly in their C-terminal regions.

Heterologous overexpression and purification of four common subunits of nuclear RNA polymerases I, II and III of Schizosaccharomyces pombe.

Four subunits of Schizosaccharomyces pombe RNA polymerases I-III shared by all three enzymes (Rpb5, Rpb8, Rpb10 and Rpc10 [Rpb12]) have been overexpressed in Escherichia coli expression vectors pQE or pET as hexahistidine fusions. The recombinant proteins have been purified to near homogeneity using metal-chelate affinity chromatography and gel filtration. Homogeneity and identity of the purified protein preparations was demonstrated by denaturing polyacrylamide gel electrophoresis and TOF-MALDI mass spectrometry.

The genome sequence of Schizosaccharomyces pombe.

We have sequenced and annotated the genome of fission yeast (Schizosaccharomyces pombe), which contains the smallest number of protein-coding genes yet recorded for a eukaryote: 4,824. The centromeres are between 35 and 110 kilobases (kb) and contain related repeats including a highly conserved 1.8-kb element.

A human RNA polymerase II subunit is encoded by a recently generated multigene family.

Background: The sequences encoding the yeast RNA polymerase II (RPB) subunits are single copy genes.

Results: While those characterized so far for the human (h) RPB are also unique, we show that hRPB subunit 11 (hRPB11) is encoded by a multigene family, mapping on chromosome 7 at loci p12, q11.23 and q22.

Partners of Rpb8p, a small subunit shared by yeast RNA polymerases I, II and III.

Rpb8p, a subunit common to the three yeast RNA polymerases, is conserved among eukaryotes and absent from noneukaryotes. Defective mutants were found at an invariant GGLLM motif and at two other highly conserved amino acids. With one exception, they are clustered on the Rpb8p structure.

Functional conservation of RNA polymerase II in fission and budding yeasts.

The complementary DNAs of the 12 subunits of fission yeast (Schizosaccharomyces pombe) RNA polymerase II were expressed from strong promoters in Saccharomyces cerevisiae and tested for heterospecific complementation by monitoring their ability to replace in vivo the null mutants of the corresponding host genes. Rpb1 and Rpb2, the two largest subunits and Rpb8, a small subunit shared by all three polymerases, failed to support growth in S. cerevisiae.

Rpc19 and Rpc40, two alpha-like subunits shared by nuclear RNA polymerases I and III, are interchangeable between the fission and budding yeasts.

The cDNAs and genes encoding the common subunits Rpc19 and Rpc40 of nuclear RNA polymerases I and III of Schizosaccharomyces pombe were isolated from cDNA and genomic libraries of the fission yeast and tested for their ability to substitute for the homologous genes in Saccharomyces cerevisiae by heterospecific complementation of corresponding null alleles and temperature-sensitive mutations. The results obtained indicate that both Sz. pombe genes (rpc19(+) and rpc40(+)) are able to replace their S.

Interactions between the full complement of human RNA polymerase II subunits.

As an approach to elucidating the rules governing the assembly of human RNA polymerase II (hRPB), interactions between its subunits have been systematically analyzed. Eleven of the 12 expected hRPB subunits have previously been tested for reciprocal interactions (J. Biol.

Mutants in ABC10beta, a conserved subunit shared by all three yeast RNA polymerases, specifically affect RNA polymerase I assembly.

ABC10beta, a small polypeptide common to the three yeast RNA polymerases, has close homology to the N subunit of the archaeal enzyme and is remotely related to the smallest subunit of vaccinial RNA polymerase. The eucaryotic, archaeal, and viral polypeptides share an invariant motif CX2C. CC that is strictly essential for yeast growth, as shown by site-directed mutagenesis, whereas the rest of the ABC10beta sequence is fairly tolerant to amino acid replacements.

Four subunits that are shared by the three classes of RNA polymerase are functionally interchangeable between Homo sapiens and Saccharomyces cerevisiae.

Four cDNAs encoding human polypeptides hRPB7.0, hRPB7.6, hRPB17, and hRPB14.

The fission yeast Schizosaccharomyces pombe rpb6 gene encodes the common phosphorylated subunit of RNA polymerase and complements a mutation in the corresponding gene of Saccharomyces cerevisiae.

A single-copy gene, homologous to the RPB6 gene from Saccharomyces cerevisiae, encoding a small phosphorylated subunit common to all three forms of nuclear DNA-dependent RNA polymerase was isolated from the fission yeast Schizosaccharomyces pombe. Its cDNA copy consists of an open reading frame of 142 codons and encodes an acidic protein (predicted pI 4.1) with a M(r) of 15,730.

Sequence analysis of closely related retrotransposon families from fission yeast.

Two families of retrotransposons, Tf1 and Tf2, have been isolated from the fission yeast, Schizosaccharomyces pombe. We report here the nucleotide (nt) sequence of a Tf2 element, the only retrotransposon family known from the commonly used laboratory strains, 972 and 975, and their derivatives. The total nt sequence of Tf2 was derived from the complete sequence of the coding region and 3' long terminal repeat (LTR) of randomly cloned element Tf2-1, and from a full 5' LTR and approximately one-third of the open reading frame (ORF) of Tf2-43, a Tf2 element found in the head-to-head orientation adjacent to the Sz.

Lambda plac10 transducing bacteriophage: DNA primary structure of the region of the abnormal excision.

In studying molecular mechanisms of the formation of transducing bacteriophages, we have elucidated the primary structure of the phage-bacterial DNA junction which resulted from the abnormal excision of the lambda plac10 phage. The process is structurally similar to the excision of the lambda plac5 phage and involves, in both cases, highly homological DNA stretches approximately 20 bp long, one of them being a part of the Z-Y spacer of the lac operon and possessing a developed secondary structure. The conception of regioselective recombination as a type of illegitimate recombinational process with a certain degree of site-specificity is suggested.

Structural bases of a long-stretched deletion: completing the lambda plac5 DNA primary structure.

In studying molecular mechanisms of specialised transduction, the lacI (E. coli)-Ea47 (lambda) DNA junction in transducing bacteriophage lambda plac 5 has been structurally elucidated, thus yielding the complete sequence of lambda plac 5 DNA including the lac5 substitution, a well-known segment of lambdoid vectors. The lambda plac5 DNA is shown to consist of 19368 bp (lambda left arm) + 3924 bp (lac5 substitution) + 25353 bp (lambda right arm), totally amounting to 48645 bp.

Site-specificity of abnormal excision: the mechanism of formation of a specialized transducing bacteriophage lambda plac5.

Molecular mechanism of the specialized transducing bacteriophage lambda plac5 formation has been studied. Phage-bacterial DNA junctions in lambda plac5 DNA are localized and primary structure of regions of the abnormal excisional recombination leading to the phage formation is elucidated; the crossover region proved to be comparable with the central part of attP and attB sites (the core and the adjacent tetranucleotide) in length and degree of homology. Bacterial insert in lambda plac5 DNA is shown to end immediately after Z-Y spacer, the DNA not containing lacY gene segments.