Characterization of inverted repeated sequences in wheat nuclear DNA.

The properties of inverted repeated sequences in wheat nuclear DNA have been studied by HAP(1) chromatography, nuclease S1 digestion and electron microscopy. Inverted repeated sequences comprise 1.7% of wheat genome. The HAP studies show that the amount of "foldback HAP bound DNA" depends on DNA length. Inverted repeats appear to be clustered with an average intercluster distance of 25 kb. It is estimated that there are approximately 3 x 10(6) inverted repeats per haploid wheat genome. The sequences around inverted repeats involve all families of repetition frequencies. Inverted repeats are observed as hairpins in electron microscopy. 20% of hairpins are terminated by a single-stranded spacer ranging from 0.3 to 1.5 kb in length. Duplex regions of the inverted repeats range from 0.1 to 0.45 kb with number average values of 0.24 kb and 0.18 kb for unlooped and looped hairpin respectively. Thermal denaturations and nuclease S1 digestions have revealed a length of about 100 bases for duplex regions. The methods used to study inverted repeated sequences are compared and discussed.     

Mapping sequences in loops of nuclear DNA by their progressive detachment from the nuclear cage.

Nuclear DNA is organised into loops, probably by attachment to a supramolecular structure. We describe a method which enables us to map the position of sequences within a loop relative to the point of attachment. Nuclear DNA is isolated unbroken by lysing HeLa cells in 2M NaCl to release structures which retain many of the morphological features of nuclei. Their DNA is supercoiled and so must remain unbroken and looped during lysis. Nucleoids are digested to various degrees with a restriction endonuclease and the cages - and any associated DNA - sedimented free from unattached DNA. The cage-associated DNA is purified and completely fragmented using the same restriction endonuclease. Equal weights of fragmented DNA are separated by gel electrophoresis, transferred to a filter and the relative amounts of the alpha, beta and gamma globin genes on the filter determined by hybridisation to the appropriate probes. The alpha genes, unlike the beta and gamma genes, resist detachment from the cage and so must lie close to the point of attachment to the cage. Our ability to map these genes implies that sequences cannot be attached at random to the cage; rather, specific sequences must be attached, so looping the DNA.     

Characterization of inverted repeated sequences in Ascaris nuclear DNA

The inverted repeated sequences of the chromatin-eliminating nematode Ascaris lumbricoides var. suum have been examined by electron microscopy and by hydroxyapatite chromatography, both in the germ-line and in the somatic DNA. 38% of the inverted repeats of the germ-line DNA analysed in the electron microscope have a single-stranded loop, in comparison to about 50% of looped structures in the somatic DNA. The loops are on average 2.3 X 10(3) base pairs (bp) long. The rest of the foldback DNA consists of simple hairpins. The average length of looped and unlooped inverted repeats is of the order of 300-400 bp in the germ-line and in the somatic DNA. The content of S1-resistant foldback duplexes isolated by hydroxyapatite chromatography amounts to 1.3% in spermatids, with an average length of 350 bp, and to 1.1% in intestinal or larval cell nuclei, with a length of about 320 bp. We estimate by two different methods that there exist approximately 12500 inverted repeats per haploid germ-line genome and approximately 8000 in the haploid somatic genome. A statistical analysis of the data indicates that the great majority of the foldback sequences are randomly distributed in the Ascaris genome, with a spacing of about (40-80) X 10(3) bp, both in the germ-line and in the somatic DNA.     

Removal of repeated sequences from hybridisation probes.

Pre-reassociation of human clone probes, containing dispersed highly repeated sequences, (e.g. Alu and KpnI families), with a large excess of sonicated total human DNA allows signal from single and low copy number components to be detected in transfer hybridisations. The signal from non-dispersed repeated sequences is reduced to single copy levels. The procedure, which is simple and quick, is illustrated using model combinations of well characterised cloned probes, and is applied to a sample of randomly chosen cosmid clones. A theoretical assessment is presented which may be useful to those wishing to use this procedure.     

Highly repeated DNA of the baboon: organization of sequences homologous to highly repeated DNA of the African green monkey.

In the African green monkey genome, 20% of the total DNA consists of a highly reiterated DNA sequence that occurs largely in long tandem arrays of a repeat unit that is 172 base-pairs in length. The DNA of the baboon contains sequences homologous to this repeat unit. However, in the baboon genome, these sequences comprise roughly 6% of the total DNA and alternate in a regular fashion with a DNA segment that may be distantly related to the monkey repeat unit. The sequences in the baboon that are homologous to the monkey repeat unit are contained within a 340 base-pair repeat unit of the highly repeated DNA fraction of the baboon. The extent of nucleotide divergence of the homologous repeated sequences between the two species is estimated to be about 10%.     

Attachment of muscle filaments to the outer membrane of the nuclear envelope

Summary Myofilaments of striated muscles can be recognized in the electron microscope to be in structural continuity with the outer membrane of the nuclear envelope. The very site of insertion of these myofilaments at the membrane surface frequently appears characterized by a dense basal knob of 85–135 Å. It is hypothesized that this attachment of myofilaments to the nuclear membrane plays a role in mechanically transmitting the contraction of the fiber to the nucleus, thus bringing about the harmonica-like folded appearance of the nucleus which is known for the contracted states of striated, smooth and cardiac muscles.The work was supported in part by the Deutsche Forschungsgemeinschaft.The author is indebted to Miss Sigrid Krien and Miss Marianne Winter for careful technical assistance as well as to Drs. Heinz Falk and U. Scheer for valuable discussions.     

Internally repeated sequences in ras gene products.

The preparation of peptides terminating in -Arg-CHN2 has been attempted because of their potential value as proteinase inactivators. We have succeeded in one case, converting Cbz-Phe-ArgOH to the diazomethane without blocking the guanidino group. As expected from previous results with such reagents, the new derivative was extremely effective in inactivating a cysteine proteinase specific for cleaving at arginyl bonds, that is, clostripain. However, in contrast with the inertness of serine proteinases to reagents of this type in the cases examined previously, plasma kallikrein was inactivated by Cbz-Phe-Arg-CHN2, although with a considerably lower rate constant than with clostripain. Trypsin, however, was not inactivated, but gradually destroyed the reagent, as had been observed previously with chymotrypsin and Cbz-Phe-CHN2. This has now been re-examined with rho-nitro-Cbz-Ala-Phe-CHN2 and shown to involve a cleavage to rho-nitro-Cbz-Ala-PheOH, probably with liberation of diazomethane.     

Simple repeated sequences in human satellite DNA.

In an extensive analysis, using a range of restriction endonucleases, HinfI and TaqI were found to differentiate satellites I, II and III & IV. Satellite I is resistant to digestion by TaqI, but is cleaved by HinfI to yield three major fragments of approximate size 770, 850 and 950bp, associated in a single length of DNA. The 770bp fragment contains recognition sites for a number of other enzymes, whereas the 850 and 950bp fragments are "silent" by restriction enzyme analysis. Satellite II is digested by HinfI into a large number of very small (10-80bp) fragments, many of which also contain TaqI sites. A proportion of the HinfI sites in satellite II have the sequence 5'GA(GC)TC. The HinfI digestion products of satellites III and IV form a complete ladder, stretching from 15bp or less to more than 250bp, with adjacent multimers separated by an increment of 5bp. The ladder fragments do not contain TaqI sites and all HinfI sites have the sequence 5'GA(AT)TC. Three fragments from the HinfI ladder of satellite III have been sequenced, and all consist of a tandemly repeated 5bp sequence, 5'TTCCA, with a non-repeated, G+C rich sequence, 9bp in length, at the 3' end.     

A high frequency of length polymorphisms in repeated sequences adjacent to Alu sequences

We describe a new class of DNA length polymorphism that is due to a variation in the number of tandem repeats associated with Alu sequences (Alu sequence-related polymorphisms). The polymerase chain reaction was used to selectively amplify a (TTA)n repeat identified in the 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase gene from genomic DNA of 41 human subjects, and the size of the amplified products was determined by gel electrophoresis. Seven alleles were found that differed in size by integrals of three nucleotides. The allele frequencies ranged from 1.5% to 52%, and the overall heterozygosity index was 62%. The polymorphic TTA repeat was located adjacent to a repetitive sequence of the Alu family. A homology search of human genomic DNA sequences for the trinucleotide TTA (at least five members in length) revealed tandem repeats in six other genes. Three of the six (TTA)n repeats were located adjacent to Alu sequences, and two of the three (in the genes for beta-tubulin and interleukin-1 alpha) were found to be polymorphic in length. Tandemly repetitive sequences found in association with Alu sequences may be frequent sites of length polymorphism that can be used as genetic markers for gene mapping or linkage analysis.     

Tandemly repeated pentanucleotides in DNA sequences of eucaryotes.

Genetic sequence data banks were scanned in order to retrieve tandemly repeated pentanucleotides (pnts). It was found that among 102 (=(1024-4)/2/5) possible distinct pnts roughly each fourth is involved in tandem repeats. It is shown that tandemly repeated pnts are composed of frequently occurring di- and trinucleotides and that those pnts which occur frequently in the form of mono- or di-pnts form also tandem repeats either in the form of satellites or in the form of shorter tandem repeats. Human satellite III is taken as a specific example. It is shown that the first guanine within GG-AAT pnt exhibits the highest mutability. Sequential distribution of base changes gives evidence that the mutations do not occur at random positions but in a correlated fashion so that long stretches of original pnts remain intact. It is found that pnts related to the satellite III are present in introns and flanking regions of some structural genes, but are not preserved between orthologous genes of related species. The results corroborate the most plausible mechanism of their evolution--rapid amplification followed by successive divergence of repeat units by various mutational processes.     

Ribosomal DNA sequences attached to the nuclear matrix

The organization of rat liver ribosomal DNA (rDNA) as matrix-attached DNA loops was examined using a protocol which fractionates chromatin from discrete regions of DNA loops. Southern blot analysis of matrix-attached and solubilized chromatin DNA fragments demonstrated that rDNA is associated with the matrix via its 5' and 3' nontranscribed spacer sequences (NTS). Although the 45 S rRNA coding sequences were approximately threefold enriched in matrix preparations, the recovery of this DNA (unlike the NTS) was dependent on the extent of nuclease digest and proportional to the length of the matrix-attached DNA fragments. The data suggest that rDNA is organized as matrix-attached DNA loops and only the NTS are directly involved in matrix binding. Further, we demonstrated that while the kinetics and extent of nuclease digestion were similar in all regions of the DNA loops, the nuclease digestion pattern of bulk nuclear and matrix DNA showed a typical nucleosome organization, but the rDNA fragments retained with the nuclear matrix did not.     

Human DNA sequences complementary to the small nuclear RNA U2.

Clones containing sequences complementary to the small nuclear RNA U2 were isolated from a human DNA library (1). Three clones, designated U2/4, U2/6 and U2/7 were purified and characterized by restriction enzyme cleavage, hybridization and heteroduplex analysis. Hybridization showed that the three clones each contained one single region which is complementary to U2 RNA. Restriction enzyme cleavage revealed furthermore that the inserted fragments in the three recombinants are different. Heteroduplex analysis identified a 240-380 bp long duplex region in each heteroduplex which includes sequences complementary to U2 RNA. Heteroduplexes between clones U2/4 and U2/7 as well as between U2/4 and U2/6 revealed two additional approximately 200 bp long homologies. The remainder of the inserts were found to lack measurable sequence homology. Two fragments from clone U2/4 were subcloned in the pBR322 vector and the subclones were used to determine the nucleotide sequence of a region in clone U2/4 which is complementary to U2 RNA. A comparison between the established sequence and the sequence for rat U2 RNA (2) reveals several discrepancies.     

Attachment of origins of replication to the nuclear matrix and the chromosomal scaffold

We have investigated the attachment of DNA to the nuclear matrix and chromosomal scaffold in synchronized bovine liver cells. Label incorporated at the onset of the S phase remained preferentially associated with the matrix during the subsequent G1 phase and with a residual protein structure from dehistonized chromosomes during mitosis. On the other hand label incorporated during mid or late S phase was about equally distributed over the DNA molecule after a chase into the G1 phase. These results suggest that DNA is attached to the nuclear matrix and chromosome scaffolds by the origins of replication.     

Interspersion and transcription of repeated sequences of rat DNA.

Repetitive rat DNA reassociated to Cot=0.1 and deprived of "foldback" sequences showed close interspersion with unique sequences. As measured by electron microscopy, the average length of repetitive segments was about 600 +/- 400, and of unique segments 1800-3600 base pairs. Pyrimidine tracts over 80 nucleotides in length were found mainly in foldback and repetitive fractions. Oligo(dT) tracts, 20-30 bases in length prevailed in the DNA fraction reassociated to Cot=0.1. Repetitive and unique DNA fractions were annealed to Millipore filters and hybridized with hnRNA. Up to 1.6% of repetitive DNA reassociated to Cot=0.05 showed base complementarity with hnRNA, whereas the comparative figures for DNA reassociated to Cot=10 and for the unique fraction were 0.8% and 0.3% respectively. When hybridization of hnRNA was carried out in solution in vast DNA excess, no hybrid formation with repetitive sequences reassociated to Cot=0.1 was observed, although hybridization with DNA reassociated to Cot=10 was noticeable.     

Long interspersed repeated sequences of the mouse genome

Long interspersed repeated sequences of the mouse genome can be prepared by digesting reassociated DNA with single-strand nuclease. Length resolution reveals many discrete bands that can be assigned to 15 kbp and 6 kbp groups. The reassociated 6 kbp group (which we identify with the MIF-1 family) possesses significant sequence heterogeneity, evidenced by the production of several smaller fragments upon single-strand nuclease digestion of heteroduplexes. The sites of sequence heterogeneity are relatively few and can be mapped using additional restriction endonuclease cuts. We have mapped additional restriction sites into this group, particularly within a cloned HindIII 400 bp fragment, and have also clearly mapped one end of this relatively homogeneous long interspersed repeated sequence.     

Characterization of species-specific repeated DNA sequences from B. nigra

Summary The construction and characterization of two genome-specific recombinant DNA clones from B. nigra are described. Southern analysis showed that the two clones belong to a dispersed repeat family. They differ from each other in their length, distribution and sequence, though the average GC content is nearly the same (45%). These B genome-specific repeats have been used to analyse the phylogenetic relationships between cultivated and wild species of the family Brassicaceae.     

Multi-site binding of human nuclear protein to the Alu-family repeated DNA

Nuclear protein which selectively binds to the Alu-family DNA repeat (AFR, Blur8) is partially purified from human HeLa cells using a gel retention assay. At low protein concentrations only a single complex of the protein with AFR is formed (CII). Increasing protein concentrations lead to the gradual disappearance of CII, being replaced by complexes with higher (CI) and lower (CIII, CIV) electrophoretic mobilities. Differential binding of AFR restriction subfragments indicates that multiple protein-binding sites are present within AFR. We discuss two models explaining the anomalous electrophoretic mobility of CII by DNA bending or looping upon cooperative multi-site binding of the protein to AFR.     

Specific nucleotide sequences in HeLa cell inverted repeated DNA: enrichment for sequences found in double-stranded regions of heterogeneous nuclear RNA

Inverted repeat DNA was isolated from HeLa cell nuclei and transcribed in vitro with Escherichia coli RNA polymerase in the presence of [alpha-³²P]nucleoside triphosphates. The RNA products were digested with T₁ ribonuclease and subjected to separation in two dimensions. The pattern of the prominent oligonucleotides was almost indistinguishable from that seen when the double-stranded regions from ³²P-labeled HeLa cell heterogeneous nuclear RNA were fingerprinted in a similar manner. The sequences of several of the largest prominent T₁ ribonuclease-generated oligonucleotides were determined and were found to agree with those isolated from the double-stranded heterogeneous nuclear RNA that migrated to the same positions in the fingerprints. The most prominent component of the inverted repeat DNA appears to be sequences that are transcribed into double-stranded regions in heterogeneous nuclear RNA molecules.