Nested retrotransposons in the intergenic regions of the maize genome.

The relative organization of genes and repetitive DNAs in complex eukaryotic genomes is not well understood. Diagnostic sequencing indicated that a 280-kilobase region containing the maize Adh1-F and u22 genes is composed primarily of retrotransposons inserted within each other. Ten retroelement families were discovered, with reiteration frequencies ranging from 10 to 30,000 copies per haploid genome. These retrotransposons accounted for more than 60 percent of the Adh1-F region and at least 50 percent of the nuclear DNA of maize. These elements were largely intact and are dispersed throughout the gene-containing regions of the maize genome.

An evolutionarily conserved protein fraction stably linked to DNA.

Chromatins from four evolutionarily remote species (insect, fish, amphibian and bird) were isolated, high-salt-extracted and extensively deproteinized to remove noncovalently associated proteins. A protein fraction resisting the extraction procedures was found firmly linked to DNA in all four chromatins. Two-dimensional tryptic peptide mapping revealed a remarkable evolutionary conservativeness of this protein component, suggesting an indispensable function for it in the nucleus.

Stable DNA-protein complexes in eukaryotic chromatin.

Demembranized sperm and somatic nuclei of mammalian origin were extracted with high salt/urea/2-mercaptoethanol, treated with detergents and purified in CsCl density gradients to isolate DNA. Under these conditions a protein component still remained bound to DNA. This stable DNA-protein complex could be reduced to an oligodeoxynucleotide-peptide complex by extensive sequential digestions with DNase I and Pronase E. Chemical and enzymatic treatments of this complex indicated the presence of a phosphoester bond between DNA and a hydroxyamino acid. Two-dimensional tryptic peptide mapping revealed a remarkable similarity among the covalently linked protein components in all types of chromatin studied. These maps differed from the maps of mammalian topoisomerases I and II.

Nonprotamine nucleoprotein ultrastructures in mature ram sperm nuclei.

Nuclei from mature ram spermatozoa were treated with a solution of 0.25 M 2-mercaptoethanol, 2 M NaCl, 4 to 8 M urea to dissociate protamines and some other proteins from DNA. The material remaining after such a treatment was spread for electron microscopy in the microcentrifugation chamber. Two types of structures were observed determined by proteins of nonprotamine nature. The first type was represented by protein bodies of an irregular ringlike shape to which DNA fibers were anchored to form a network. This structure determines the shape of the sperm head and may correspond to the nuclear skeleton described in somatic cells. The second type of structures were chromatin fibers containing beads of approximately nucleosomal size. These rough fibers were unevenly distributed in the nucleus and were much less frequent than the smooth fibers usually observed. Both types of structures were determined by unusually firmly bound proteins. They were resistant not only to the reduction of disulfide bonds and to high salt and urea concentrations but also to 2% sodium dodecysulfate and to 5 M guanidine chloride. These results show that apart from packing of DNA in a nucleoprotamine complex, two levels of DNA organization can be observed in the ram sperm nucleus: the first level consists of two kinds of DNA fibers (smooth and rough); in the second level of DNA is organized in domains fixed by a proteinous nuclear skeleton.

Trout sperm chromatin. II. Ultrastructural aspects after salt dissociation of proteins.

The ultrastructural organization of the trout sperm nucleus was studied in ultrathin sections and spread preparations after partial decondensation of the nucleus with increasing NaCl concentrations. The obtained results suggest that the organization of the trout sperm chromatin is much more complex than a pure nucleoprotamine. Three types of complexes were observed. The first one results from the association of DNA with protamines. This complex appears as a fibrous network when partially decondensed nuclei are digested with DNase I indicating that at least a part of DNA remains protected by protamines and favours models accepting a colinear alignment of the latter on the DNA molecules. The second type of structures represent the DNA-protamine fibers compacted into dense clumps which appear as separate compaction units seen upon partial decondensation of the sperm nucleus. A third type are complexes of the ring-shaped granular bodies tightly associated with DNA and resisting high salt-urea and detergent treatment.

Trout sperm chromatin. I. Biochemical and immunological study of the protein composition.

Compact sperm chromatin was obtained from mature trout sperm nuclei resistant to sonication and detergent treatments. 0.5 to 2 M NaCl caused a gradual decondensation of this chromatin and the dependence of the percentage of dissociated proteins on the salt concentration indicated cooperativity of the dissociation process. Urea alone was insufficient to decondense the nuclei. The only proteins dissociated from the sperm nuclei by NaCl alone or combined with urea were protamines. Besides protamines, tightly bound nonprotamine proteins resisting high salt-urea extraction were detected in the sperm nucleus. Part of them could be solubilized by 1% sodium dodecyl sulphate (SDS) and displayed the characteristics of the core histones: they were soluble in 0.25 N H2SO4, their electrophoretic mobilities were similar to those of trout liver core histones, and they shared common antigenic determinants with the latter. The rest of the tightly bound proteins resisted 1% SDS treatment and could be obtained after an extensive digestion of DNA with DNase I. These were nonhistone proteins similar in mobility to the protein triplet characteristic of the lamina-pore complex and an additional high molecular weight protein.

Two Arabidopsis homologs of the animal trithorax genes: a new structural domain is a signature feature of the trithorax gene family.

Two Arabidopsis genes have been characterized as first examples of plant genes homologous to the animal trithorax genes. The Arabidopsis genes are highly similar but display different tissue and development expression patterns. One of them was ubiquitously expressed, with highest levels registered in young seedlings. The other gene was less active in all tested tissues, was not expressed in mature leaves but was highly expressed in roots. A new structural motif common to all TRX-related proteins has been identified. This new architectural element was found only in genes of multicellular species and is present in all genes belonging to the trithorax family. Along with the SET domain and the PHD fingers, this new element is a signature feature for the trithorax gene family.

DNA sequences tightly bound to proteins in mouse chromatin: identification of murine MER sequences.

The finding of stably (tightly) associated DNA-protein complexes in eukaryotic chromatin has provoked many hypotheses and speculations concerning their possible role. While the answer of this question is not envisaged yet, it is clear that elucidation of the nature of the individual components involved in such complexes is a necessary step in this direction. Here, the nature of several mouse DNA sequences in the vicinity of a putative stably attached protein is studied. Eight independently isolated clones containing such sequences were compared to known sequences in GenBank. Two clones were found to belong to different subfamilies of repetitive sequences, organized into a larger family--the L1md family. One clone harbors a sequence that is a member of the Alu-type family. Four of the cloned sequences are preset in low copy numbers, but the computer search found similar sequences in various genomic regions of different rodents. These facts, together with the finding that regions homologous to the above clones often flank other repetitive elements in the genome, suggest that the cloned sequences belong to new, not yet described families of repeats in the murine genome. It is possible that they correspond to the medium reiteration frequency sequences, MER-sequences, discovered recently in the human genome (Jurka, 1990; Kaplan and Duncan, 1990). Particularly intriguing is the homology found at the integration sites of polyoma virus in two transformed cell lines with two of these clones.

[Role of the ribose residue of substrates in reactions catalyzed by ribonuclease]. / Rol' riboznogo ostatka substratov v reaktsiiakh, kataliziruemykh ribonukleazami.

The rate constants and Km for the hydrolysis of the optically active nonglycosidic analogues of the CpA and C greater than p catalysed by RNase A and RNase BS-I were measured. The rate of hydrolysis of the model substrates in 10(5) and 10(3) slower that for the appropriate dinucleoside phosphate and nucleoside cyclophosphate. However, substitution of the relatively rigid ribofuranose ring with flexible alifatic chains is accompanied by little variation in binding constants. The analyses based on the single substrate system indicate that the observed difference in rate constants must be accounted for by a difference between the binding of the substrates in the transition state to the RNase active site. Consequently, the "rigidity" of the ribose rings in RNA leads to large decreases in the free energy of activation for the reactions catalysed by RNases.

[Conformational stability of ribonuclease A complexes with specific inhibitors]. / Konformatsionnaia stabil'nost' kompleksov ribonukleazy A so spetsificheskimi ingibitorami.

Isotermic unfolding of ribonuclease A, phosphopyridoxyl-8Lys41-RNAase A and complexes of the enzyme with cytidine, 2'-CMP, 3'-CMP, 3'-AMP and with the phosphoric ester of 1-(omega-oxypropyl)-cytosine in presence of urea has been studied. The stabilization of the protein structure resulting from the complex formation was shown to be determined by the ligand nucleobase binding. The comparison of the results obtained with those known from the literature suggests, that binding and catalytic zones of the enzyme active site form an integrated network system which is substained by multipoint contacts between the constituents. The change in the state of any part within the enzyme active state affects the energetics of the whole protein globule.

Stably DNA-bound chromosomal proteins.

DNA accomplishes its biological function in a complex with nuclear proteins. A minor protein fraction has been found in chromatin which could not be dissociated from DNA by reagents abolishing non-covalent type of interactions. The controversy surrounding the nature of the protein moiety and the nature of the bond linking the two components on the one hand, and the fact pointing to its evolutionary conservatism and metabolic stability on the other, make it necessary to critically evaluate the data in view of the possible biological function for such proteins.

Interaction of MAR-sequences with nuclear matrix proteins.

The recent discovery of DNA sequences responsible for the specific attachment of chromosomal DNA to the nuclear skeleton (MARs/SARs) was an important step towards our understanding of the functional and structural organization of eukaryotic chromatin [Mirkovitch et al.: Cell 44:273-282, 1984; Cockerill and Garrard: Cell 44:273-282, 1986]. A most important question, however, remains the nature of the matrix proteins involved in the specific binding of the MARs. It has been shown that topoisomerase II and histone H1 were capable of a specific interaction with SARs by the formation of precipitable complexes [Adachi et al.: EMBO J8:3997-4006, 1989; Izaurralde et al.: J Mol Biol 210:573-585, 1989]. Here, applying a different approach, we were able to "visualize" some of the skeletal proteins recognizing and specifically binding MAR-sequences. It is shown that the major matrix proteins are practically the same in both salt- and LIS-extracted matrices. However, the relative MAR-binding activity of the individual protein components may be different, depending on the method of matrix preparation. The immunological approach applied here allowed us to identify some of the individual MAR-binding matrix proteins. Histone H1 and nuclear actin are shown to be not only important components of the matrix, but to be involved in a highly efficient interaction with MAR-sequences as well. Evidence is presented that proteins recognized by the anti-HMG antibodies also participate in MAR-interactions.

Tightly bound nonprotamine proteins from ram sperm nuclei studied by one- and two-dimensional peptide mapping.

The tightly bound proteins of ram sperm nuclei (TBSP) have been recovered as a fraction co-sedimenting with DNA after high salt-urea deprotamination of the nuclei. TBSP were studied by two-dimensional (2D)-tryptic peptide mapping and by one-dimensional (1D)-partial proteolysis mapping. The 2D maps revealed a strong homology among the proteins, irrespective of substantial differences in their molecular masses. This homology was supported also by the 1D-mapping data. The 2D-tryptic maps of TBSP were compared to those of lamb liver lamins but no apparent similarity was detected. TBSP were found to react positively to a test for the presence of carbohydrate residues, suggesting that these proteins are glycoproteins as established earlier for the lamins. The 2D maps of several proteins of seminal plasma origin, used as a control, displayed completely different peptide profiles.

Matrix attachment sites in the murine alpha-globin gene.

DNA sequences with a high affinity for nuclear matrix proteins have been identified and localized in the mouse alpha-globin gene. These matrix association regions (MARs) are adjacent, covering the first intron and part of the 5'-coding sequence. The binding sites are in close proximity to DNase I hypersensitive sites and other important signal sequences. The proteins of the nuclear lamina do not bind the alpha-globin gene MARs in the in vitro binding assay. The finding of MARs in the mouse alpha-globin gene creates an apparent paradox, since works from other authors and our results presented here indicate that this gene is not bound to the nuclear matrix in vivo. This contradiction is difficult to explain at present but different possibilities are accounted for in the text.

Isolation of matrices from maize leaf nuclei: identification of a matrix-binding site adjacent to the Adh1 gene.

Nuclear matrices were isolated from maize leaves by the two conventional methods usually employed for the preparation of the corresponding structures of animal origin. It is demonstrated that functionally competent matrices, recognizing and specifically binding the MAR-containing DNA of the mouse kappa-immunoglobulin gene may be prepared by both 2 M NaCl and LIS extractions of maize nuclei. A DNA region with a high affinity for the nuclear matrix was identified at the 5' end of the maize Adh1-S gene, distal to the promoter region. The presence of sites of reported altered chromatin structure in this particular region is discussed. While the proximity and the cohabitation of MARs with different regulatory elements is a common feature of matrix association regions in animal systems, this is the first plant MAR identified in a region of known significance for gene regulation.

Binding of sequences from the 5'- and 3'-nontranscribed spacers of the rat rDNA locus to the nucleolar matrix.

Nucleolar matrix structures were obtained under different extraction conditions from highly purified isolated nucleoli. Their ultrastructural appearance, protein composition and capacity to bind rDNA preferentially were studied in a model binding system. A region spanning approximately 25 kb in the rat ribosomal gene locus was screened for DNA sites capable of specifically interacting with the proteins of the nucleolar matrix (MARs). Two such sites were identified: one is located on an EcoRV-KpnI fragment in the 5'-nontranscribed spacer region, between two repetitive elements and close to the transcription initiation site; the other MAR is on a PvuII-BamHI fragment located in the 3'-nontranscribed region, encompassing an element 85% homologous to a B2-sequence. The two MARs are located in regions rich in polypyrimidine/polypurine tracks and contain a few elements homologous to the consensus sequence for topoisomerase II. This indicates that the "attachment sites" for the ribosomal genes belong to the same class of sequences as the MARs attaching the chromosomal DNA to the nuclear matrix.

An H1-like protein from the sperm chromatin of Mytilus galloprovincialis.

We have isolated and purified a sperm-specific protein (S3) from the mussel M. galloprovincialis. Antibodies against S3 were raised in rabbits and used for its immunological comparison to somatic histones. The results showed that S3 did not share common immunological determinants with H2b or any other core histone-contrary to the suggestion that it was an H2b-like protein (Ausio and Subirana, 1982). With H1 there was a crossreaction between S3 and anti-H1 as well as with H1 and anti-S3. Although similar to somatic H1, S3 is not identical with it. This fact makes S3 an interesting example of another protein of the H1-H5 type, present in a completely inactive chromatin.