Tightly bound to DNA proteins: possible universal substrates for intranuclear processes.

Tightly bound to DNA proteins (TBPs) are a protein group that remains attached to DNA after its deproteinization by phenol, chloroform or salting-out. TBP are bound to DNA with covalent phosphotriester or non-covalent ion and hydrogen bonds. They appear to be a vast protein group involved in numerous intranuclear processes. The TBPs fraction co-purified with DNA deproteinized by mild procedures is extremely heterogeneous, tissue and species-specific. The protein fraction co-purified with DNA after harsh deproteinization procedures appears to be formed from few polypeptides common to different species and tissues. Interaction sites between DNA and TBPs depend on the physiological status of the cell. The binding sites of TBPs to DNA do not co-localize with the nuclear matrix attachment regions. We hypothesize that TBPs form a universal substrate for intranuclear processes.

Analysis of plant diversity with retrotransposon-based molecular markers.

Retrotransposons are both major generators of genetic diversity and tools for detecting the genomic changes associated with their activity because they create large and stable insertions in the genome. After the demonstration that retrotransposons are ubiquitous, active and abundant in plant genomes, various marker systems were developed to exploit polymorphisms in retrotransposon insertion patterns. These have found applications ranging from the mapping of genes responsible for particular traits and the management of backcrossing programs to analysis of population structure and diversity of wild species. This review provides an insight into the spectrum of retrotransposon-based marker systems developed for plant species and evaluates the contributions of retrotransposon markers to the analysis of population diversity in plants.

Proteins tightly bound to DNA: new data and old problems.

Proteins tightly bound to DNA (TBP) comprise a group of proteins that remain bound to DNA after usual deproteinization procedures such as salting out and treatment with phenol or chloroform. TBP bind to DNA by covalent phosphotriester and noncovalent ionic and hydrogen bonds. Some TBP are conservative, and they are usually covalently bound to DNA. However, the TBP composition is very diverse and significantly different in different tissues and in different organisms. TBP include transcription factors, enzymes of the ubiquitin-proteasome system, phosphatases, protein kinases, serpins, and proteins of retrotransposons. Their distribution within the genome is nonrandom. However, the DNA primary structure or DNA curvatures do not define the affinity of TBP to DNA. But there are repetitive DNA sequences with which TBP interact more often. The TBP distribution within genes and chromosomes depends on a cell's physiological state, differentiation type, and stage of organism development. TBP do not interact with DNA in the sites of its association with nuclear matrix and most likely they are not components of the latter.

Possible involvement of DNA strand breaks in regulation of cell differentiation.

The present review summarizes data on the accumulation of DNA strand breaks in differentiating cells. Large 50 Kbp free DNA fragments were observed by several research teams in non-apoptotic insect, mammal and plant cells. A more intensive DNA breakage was observed during maturation of spermatides, embryo development, and differentiation of myotubes, epidermal cells, lymphocytes and neutrophils. In general, accumulation of DNA strand breaks in differentiating cells cannot be attributed to decrease of the DNA repair efficiency. Poly(ADP)ribose synthesis often follows the DNA breakage in differentiating cells. We hypothesize that DNA fragmentation is an epigenetic tool for regulation of the differentiation process. Scarce data on localization of the differentiation-associated DNA strand breaks indicate their preferred accumulation in specific DNA sequences including the nuclear matrix attachment sites and repeats. Recent data on non-apoptotic functions of caspases provide more evidence for possible existence of a DNA breakage mechanism in differentiating cells resembling the initial stage of apoptosis. Excision of methylated cytosine and recombination are other possible explanations of the phenomenon. Elucidation of mechanisms of differentiation-induced DNA strand breaks appears to possess considerable research potential.

[Possible involvement of DNA breaks in epigenetic regulation of cell differentiation].

The review summarizes the authors' and literature data on accumulation of DNA breaks in differentiating cells. Large 50-kb free DNA fragments were observed by several research teams in non-apoptotic insect, mammal, and plant cells. More intense DNA breakage was observed during maturation of spermatides, embryo development, and differentiation of myotubes, epidermal cells, lymphocytes, and neutrophils. In general, accumulation of DNA breaks in differentiating cells cannot be attributed to a decrease in the DNA repair efficiency. Poly(ADP)ribose synthesis often follows the DNA breakage in differentiating cells. We hypothesize that DNA fragmentation is an epigenetic tool for regulating the differentiation process. Scarce data on localization of the differentiation-associated DNA breaks indicate their preferable accumulation in specific DNA sequences including the nuclear matrix attachment sites. he same sites are degraded at early stages of apoptosis. Recent data on non-apoptotic function of caspases provide more evidence for possible existence of a DNA breakage mechanism in differentiating cells, resembling the initial stage of apoptosis. Excision of methylated cytosine and recombination are other possible explanations of the phenomenon. Elucidation of mechanisms of differentiation-induced DNA breaks appears to be a prospective research direction.

SNPs of PSMA6 gene--investigation of possible association with myocardial infarction and type 2 diabetes mellitus.

In our preceding studies we have identified microsatellite polymorphisms inside the PSMA6 gene and in its 5' upstream region. Following the observed associations of microsatellite polymorphisms with non-insulin dependent diabetes mellitus and Graves' disease we extended the evaluation of PSMA6 genetic variations to cardiovascular disorders and non-insulin dependent diabetes mellitus. New polymorphisms in the promoter region and exon 6 of the gene were identified by direct sequencing of the promoter region and all seven exons of the gene in 30 individuals of European descent. Two SNPs at positions -110 and -8 from the translation start, in the promoter region and 5'UTR respectively, were analyzed. Neither polymorphism was associated with the risk of myocardial infarction. No significant association of the polymorphisms with plasma lipid levels or BMI was observed. A borderline association of both polymorphisms with diastolic blood pressure was observed in the control group. Genotype -8CG was significantly more frequent in type 2 diabetes patients, and haplotype C-110/G-8, compared to C-110/C-8 was associated with a higher risk of NIDDM.

Association of microsatellite polymorphisms of the human 14q13.2 region with type 2 diabetes mellitus in Latvian and Finnish populations.

A polymorphic microsatellite in intron 6 of the human proteasome core particle PSMA6 gene (HSMS006), and four other microsatellites localized upstream on human chromosome 14q13.2 (HSMS801, HSMS702, HSMS701, HSMS602), were genotyped in 104 type 2 diabetic patients and 129 age-matched control subjects from Latvia and replicated in 91 type 2 diabetic patients and 88 age-matched healthy control subjects from the Botnia Study in Finland. In type 2 diabetic patients from both populations the HSMS006 (TG)22 allele was two times more frequent compared to the control group. In the Latvian population the (CAA)8 allele of the HSMS602 marker was less frequent in the diabetic group, as was the (AC)24 allele of microsatellite HSMS801. Allele frequencies of the HSMS701 and 702 repeats were similar in healthy controls and type 2 diabetic patients. In conclusion, our data suggest that variants in the PSMA6 gene on chromosome 14q13.2 are associated with type 2 diabetes.

Novel haplotype description and structural background of the eventual functional significance of the barley beta-amylase gene intron III rearrangements.

To extend the knowledge on the haplotype variability of the Bmy1 gene, region of the intron III was sequenced in a set of 20 Latvian accessions and Danish variety Maja, the data were compared to the previously reported allelic variants of the structural gene. Taking into account the polymorphisms of 59 loci and the microsatellite (MS) motif, 11 Latvian varieties turned out to have haplotype similar to cultivar Adorra, 1 - to Haruna Nijo, and 8 - to the newly described Abava Bmy1 intron III haplotype. High level of polymorphisms of (TG)(m) as well as (G)(n) component of MS was revealed for all the haplotypes studied. We conclude that the MS motif rather than the MS size length polymorphism correlates with mutations in the coding region of the beta-amylase gene. Five graphical haplotype-specific intron III structures were constructed on the basis of the co-localization of the transcription factor binding sites (TFBSs), remnants of the transposable elements, and intron III polymorphic loci. Inter- and intrahaplotype variability was analyzed on the eventual functional significance of the Bmy1 intron III rearrangements. Novel data on the intron III nucleotide sequences of the Bmy1 gene were deposited in the GenBank (http://www.ncbi.nlm.nih.gov/) under accession numbers DQ316895-DQ316905.

Nuclear matrix proteins and hereditary diseases.

The review summarizes literature data on alterations of structure or expression of different nuclear matrix proteins in hereditary syndromes. From the point of view of involvement of nuclear matrix proteins in etiology and pathogenesis of the disease hereditary pathologies can be classified in pathologies with pathogenesis associated with defects of nuclear matrix proteins and pathologies associated to changes of the nuclear matrix protein spectrum. The first group includes laminopathies, hereditary diseases with abnormal nuclear-matrix associated proteins and triplet extension diseases associated with accumulation of abnormal proteins in the nuclear matrix. Laminopathies are hereditary diseases coupled to structural defects of the nuclear lamina. These diseases include Emery-Dreifuss muscular dystrophy, limb girdle muscular dystrophy, dilated cardiomyopathy (DCM) with conduction system disease, familial partial lipodystrophy (FPLD), autosomal recessive axonal neuropathy (Charcot-Marie-Tooth disorder type 2, CMT2), mandibuloacral dysplasia (MAD), Hutchison Gilford Progeria syndrome (HGS), Greenberg Skeletal Dysplasia, and Pelger-Huet anomaly (PHA). Most of them are due to mutations in the lamin A/C gene, one - to mutations in emerin gene, some are associated with mutations in Lamin B receptor gene. In Werner's, Bloom's, Cockayne's syndromes, Fanconi anemia, multiple carboxylase deficiency mutations in nuclear matrix protein or enzyme gene lead to deficient DNA repair, abnormal regulation of cell growth and differentiation or other specific metabolic functions. Proteins with a long polyglutamic tract synthesized in the cells of patients with dentato-rubral and pallido-luysian atrophy, myotonic dystrophy and Huntington disease interfere with transcription on the nuclear matrix. Down's syndrome is a representative of the group of diseases with altered nuclear matrix protein spectrum.

Development and genetic mapping of 127 new microsatellite markers in barley.

To enhance the marker density of existing genetic maps of barley ( Hordeum vulgare L.), a new set of microsatellite markers containing dinucleotide motifs was developed from genomic clones. Out of 254 primer pairs tested, a total of 167 primer pairs were classifed as functional in a panel of six barley cultivars and three H. spontaneum accessions, and of those, 127 primer pairs resulting in 133 loci were either mapped or located onto chromosomes. The polymorphism information content (PIC) ranged from 0.05 to 0.94 with an average of 0.60. The number of alleles per locus varied from 1 to 9. On average, 3.9 alleles per primer pair were observed. The RFLP frameworks of two previously published linkage maps were used to locate a total of 115 new microsatellite loci on at least one mapping population. The chromosomal assignment of 48 mapped loci was corroborated on a set of wheat-barley chromosome addition lines; 18 additional loci which were not polymorphic in the mapping populations were assigned to chromosomes by this method. The microsatellites were located on all seven linkage groups with four significant clusters in the centromeric regions of 2H, 3H, 6H and 7H. These newly developed microsatellites improve the density of existing barley microsatellite maps and can be used in genetic studies and breeding research.

Molecular cytogenetic analysis of wheat-barley hybrids using genomic in situ hybridization and barley microsatellite markers.

In the present investigation, genomic in situ hybridization (GISH) and barley microsatellite markers were used to analyse the genome constitution of wheat-barley hybrids from two backcross generations (BC1 and BC2). Two BC1 plants carried 3 and 6 barley chromosomes, respectively, according to GISH data. Additional chromosomal fragments were detected using microsatellites. Five BC2 plants possessed complete barley chromosomes or chromosome segments and six BC2 plants did not preserve barley genetic material. Molecular markers revealed segments of the barley genome with the size of one marker only, which probably resulted from recombination between wheat and barley chromosomes. The screening of backcrossed populations from intergeneric hybrids could be effectively conducted using both genomic in situ hybridization and molecular microsatellite markers. GISH images presented a general overview of the genome constitution of the hybrid plants, while microsatellite analysis revealed the genetic identity of the alien chromosomes and chromosomal segments introgressed. These methods were complementary and provided comprehensive information about the genomic constitution of the plants produced.

Inheritance of microsatellite alleles in pedigrees of Latvian barley varieties and related European ancestors.

Genetic diversity and inheritance of 65 microsatellite (SSR) loci were studied in a set of 37 barley varieties involved in the pedigrees of seven Latvian barley varieties: Abava, Agra, Balga, Imula, Linga, Priekulu 1 and Stendes. Cluster analysis divided all the varieties into two large groups according to their geographic distribution. Moravian, Swedish and Danish varieties clustered separately from varieties from Norway and Finland. The pattern of subgroups of both European and Latvian varieties was in accordance with their pedigree information. Graphical genotypes of microsatellite alleles of all seven barley chromosomes were determined for all the 37 varieties studied. Parental inheritance and transmission of microsatellite alleles through the generations of the pedigrees were analysed. The results confirmed the importance and informative value of microsatellite markers for genetic studies in barley and their utility for barley breeding and other applications in fundamental and applied barley genetics.

[Structure of genomic domains of mammalian and avian globin genes]. / Struktura genomnykh domenov genov globinov mlekopitaiushchikh i ptits.

The data on the genomic domain structure of both mammalian and avian alpha- and beta-globin genes are reviewed. The specific features of chromatin, DNA binding to the nuclear matrix, and domain-specific transcripts are discussed. In humans, the beta-globin gene domain is located in the GC-depleted isochore and contains multiple nuclear matrix attachment regions. The locus is controlled by six chromatin regions hypersensitive to DNase located far upstream of the first structural gene. Some of these regions display enhancer activity to support normal transcription level in the domain. Other mammalian beta-globin domains are similarly organized. The avian beta-globin genes are specifically arranged and their expression is less dependent from the locus control region. The human alpha-globin gene is located in the GC-rich isochore. The nuclear matrix attachment sites are not identified in this gene. An analog of the locus control region is located 40 kb upstream of the zeta-globin gene. The avian alpha-globin gene domains contain numerous nuclear matrix attachment regions. In these domains, an element located far upstream the genes regulates positive rather than negative transcription. An unidentified housekeeping gene as well as some other transcripts not encoding the structural globin genes is transcribed in the direction opposite to that of the globin genes in both mammalian and avian domains.

Exon/intron organisation of human proteasome PROS-27 K gene.

The exon/intron structure of the human proteasome PROS-27 gene was established by means of partial sequencing of its genomic clones and comparison with the chromosome 14 sequences from the data bases. The gene contains seven exons spanning over 19kb. Introns of the gene contain numerous Alu type repeats, Mer 2 and LINE type repeats. Pattern of the repeats indicates conservatism of the sequence.

A simple sequence repeat-based linkage map of barley.

A total of 568 new simple sequence repeat (SSR)-based markers for barley have been developed from a combination of database sequences and small insert genomic libraries enriched for a range of short simple sequence repeats. Analysis of the SSRs on 16 barley cultivars revealed variable levels of informativeness but no obvious correlation was found with SSR repeat length, motif type, or map position. Of the 568 SSRs developed, 242 were genetically mapped, 216 with 37 previously published SSRs in a single doubled-haploid population derived from the F(1) of an interspecific cross between the cultivar Lina and Hordeum spontaneum Canada Park and 26 SSRs in two other mapping populations. A total of 27 SSRs amplified multiple loci. Centromeric clustering of markers was observed in the main mapping population; however, the clustering severity was reduced in intraspecific crosses, supporting the notion that the observed marker distribution was largely a genetical effect. The mapped SSRs provide a framework for rapidly assigning chromosomal designations and polarity in future mapping programs in barley and a convenient alternative to RFLP for aligning information derived from different populations. A list of the 242 primer pairs that amplify mapped SSRs from total barley genomic DNA is presented.

A novel gene is transcribed in the chicken alpha-globin gene domain in the direction opposite to the globin genes.

A novel gene transcribed in the direction opposite to that of the globin genes was found in the chicken alpha-globin gene domain. Northern hybridisation with single-stranded riboprobes revealed that a 4.5-kb poly(A)+ RNA is transcribed in antisense polarity with respect to the globin genes. The transcription unit encoding this RNA seems to overlap the entire cluster of alpha-globin genes and extends at least 15 kb upstream from pi, the first of the alpha-globin genes. This new transcript shows partial sequence homology with that encoded by the human "-14" gene. An oligonucleotide based on part of a restriction fragment of chicken DNA that is 80% homologous to exon 4 of the human "-14" gene hybridises with a 4.5-kb RNA molecule. In situ hybridisation of globin-antisense probes, that detect polyribosomal mRNAs of 1.7 and 2.5 kb on Northern blots, shows these "antisense" transcripts to be present in the cytoplasm. The 4.5-kb RNA is absent in polyribosomal poly(A)+ RNA and may, hence, represent a nuclear pre-mRNA transcribed from the chicken gene that is homologous to the human "-14" gene. The expression of this gene is not specific to erythroid cells; analogous transcripts were also detected in poly(A)+ RNA extracted from a chicken lymphoblastoma cell line (HP50). Taken together, these data allow us to postulate the existence in the chicken genome of a novel gene, for which we suggest the name "ggPRX" in analogy to the murine mProx1, a gene identified in the upstream region of the alpha-globin gene domain in mice.