Integration site preferences of the Alu family and similar repetitive DNA sequences.

Numerous flanking nucleotide sequences from two primate interspersed repetitive DNA families have been aligned to determine the integration site preferences of each repetitive family. This analysis indicates that both the human Alu and galago Monomer families were preferentially inserted into short d(A+T)-rich regions. Moreover, both primate repeat families demonstrated an orientation specific integration with respect to dA-rich sequences within the flanking direct repeats. These observations suggest that a common mechanism exists for the insertion of many repetitive DNA families into new genomic sites. A modified mechanism for site-specific integration of primate repetitive DNA sequences is provided which requires insertion into dA-rich sequences in the genome. This model is consistent with the observed relationship between galago Type II subfamilies suggesting that they have arisen not by mere mutation but by independent integration events.     

The organization of DNA sequences in the mouse genome

Analysis of the organization of nucleotide sequences in mouse genome is carried out on total DNA at different fragment size, reannealed to intermediate value of Cot, by Ag⁺-Cs₂SO₄ density gradient centrifugation. — According to nuclease S-1 resistance and kinetic renaturation curves mouse genome appears to be made up of non-repetitive DNA (76% of total DNA), middle repetitive DNA (average repetition frequency 2×10⁴ copies, 15% of total DNA), highly repetitive DNA (8% of total DNA) and fold-back DNA (renatured density 1.701 g/ml, 1% of total DNA).— Non-repetitive sequences are intercalated with short middle repetitive sequences. One third of non-repetitive sequences is longer than 4500 nucleotides, another third is long between 1800 and 4500 nucleotides, and the remainder is shorter than 1800 nucleotides. —Middle repetitive sequences are transcribed in vivo. The majority of the transcribed repeated sequences appears to be not linked to the bulk of non-repeated sequences at a DNA size of 1800 nucleotides. — The organization of mouse genome analyzed by Ag⁺-Cs₂SO₄ density gradient of reannealed DNA appears to be substantially different than that previously observed in human genome using the same technique.     

Genome structure of Tetrahymena pyriformiis

Reassociation kinetics of DNA from the macronucleus of the ciliate, Tetrahymena pyriformis GL, has been studied. The genome size determined by the kinetic complexity of DNA was found to be 2.0×10⁸ base pairs (or 1.2×10¹¹ daltons). About 90% of the macronuclear DNA fragments 200–300 nucleotides in length reassociate at a rate corresponding to single-copy nucleotide sequences, and 7–9% at a rate corresponding to moderate repetitive sequences; 3–4% of such DNA fragments reassociate at C₀t practically equal to zero. To investigate the linear distribution of repetitive sequences, DNA fragments of high molecular weight were reassociated and reassociation products were treated with Sl-nuclease. DNA double-stranded fragments were then fractionated by size. It has been established that in the Tetrahymena genome long regions containing more than 2000 nucleotides make up about half of the DNA repetitive sequences. Another half of the DNA repetitive sequences (short DNA regions about 200–300 nucleotides long) intersperse with single-copy sequences about 1,000 nucleotides long. Thus, no more than 15% of the Tetrahymena genome is patterned on the principle of interspersing single-copy and short repetitive sequences. Most of the so called zero time binding or foldback DNA seem to be represented by inverted self-complementary (palindromic) nucleotide sequences. The conclusion has been drawn from the analysis of this fraction isolated preparatively by chromatography. About 75% of the foldback DNA is resistant to Sl-nuclease treatment. The Sl-nuclease resistance is independent of the original DNA concentration. Heat denaturation and renaturation are reversible and show both hyper and hypochromic effects. The majority of the inverted sequences are unique and about 20% are repeated tens of times. According to the equilibrium distribution in CsCl density gradients the average nucleotide content of the palindromic fraction does not differ significantly from that of total macronuclear DNA. It was shown that the largest part of this fraction of the Tetrahymena genome are not fragments of ribosomal genes.     

DNA reassociation kinetics and hybridization in different angiosperms

Reassociation kinetics ofDaucus carota andPetroselinum crispum (Apiaceae), andDatura innoxia (Solanaceae) are presented. Hybridization of³H-labelled DNA of two carrot cultivars indicate strong qualitative homologies of DNA sequences; nevertheless, certain quantitative differences in some Cotregions seem to exist. However, homologous sequences ofDaucus DNA with DNA ofDatura, and, suprisingly, even with DNA ofPetroselinum are very restricted: between 8% in the repeated regions and ca. 7–9% in the unique regions.     

Clusters of repeated sequences of chicken DNA are extensively methylated but contain specific undermethylated regions

In the chicken genome there are middle repetitive DNA sequences with a clustered organization. Each cluster is composed of members of different families of repeated DNA sequences and usually contains only one member of each family. Many clusters have the same assortment of repeated sequences but they are in scrambled order from cluster to cluster. These clusters usually exceed 20 × 10³ bases in length and comprise at least 10% of the repeated DNA of the chicken. The repeated sequences that are cluster components are extensively methylated. Methylation was detected by comparing HpaII and MspI digests of total DNA, where the occurrence of the sequence C-m⁵C-G-G is indicated when HpaII (cleaves C-C-G-G) fragments are larger than those generated by MspI (cleaves C-m⁵C-G-G or C-C-G-G). In hybridization experiments with Southern (1975) blots of total DNA digested with either HpaII or MspI, the cloned probes representing clustered repeated sequences showed a dramatic difference in the lengths of restriction fragments detected in the two digests. Many of the sequences that comprise these clusters are methylated in most of their genomic occurrences. There are patterns of methylation that are reproduced faithfully from copy to copy. The overall distribution of methylation within clusters seems to be regional, with long methylated DNA segments interrupted by specific undermethylated regions.     

An alternative view of mammalian DNA sequence organization: II. Short repetitive sequences are organized into scrambled tandem clusters in Syrian hamster DNA

Hyperchromicity, S₁ nuclease digestion, and reassociation studies of Syrian hamster repetitive DNA have led to novel conclusions about repetitive sequence organization. Re-evaluation of the hyperchromicity techniques commonly used to determine the average length of genomic repetitive DNA regions indicates that both the extent of reassociation, and the possibility of non-random elution of hyperpolymers from hydroxyapatite can radically affect the observed hyperchromicity. An alternative interpretation of hyperchromicity experiments, presented here, suggests that the average length of repetitive regions in Syrian hamster DNA must be greater than 4000 nucleotides.S₁ nuclease digestion of reassociated 3200 nucleotide Syrian hamster repetitive DNA, on the other hand, yields both long (>2000 nucleotides) and short (300 nucleotides) resistant DNA duplexes. Calculations indicate that the observed mass of short nuclease-resistant duplexes (>60%) is too large to have arisen only from independent short repetitive DNA sequences alternating with non-repetitive regions. Reassociation experiments using long and short S₁ nuclease-resistant duplexes as driver DNA indicate that all repetitive sequences are present in both fractions at approximately the same concentration. Isolated long S₁ nuclease-resistant duplexes, after denaturation, renaturation, and a second S₁ nuclease digestion, again produce both long and short DNA duplexes. Reassociation experiments indicate that all repetitive DNA sequences are still present in the “recycled” long S₁ nuclease-resistant duplexes. These experiments imply that many of the short S₁ nuclease-resistant repetitive DNA duplex regions present in reassociated Syrian hamster DNA were initially present in the genome as part of longer repetitive sequence blocks. This conclusion suggests that the majority of “short” repetitive regions in Syrian hamster DNA are organized into scrambled tandem clusters rather than being individually interspersed with non-repetitive regions.     

DNA sequence organization in the genome of Petroselinum sativum (Umbelliferae)

The genome of parsley was studied by DNA/DNA reassociation to reveal its spectrum of DNA reiteration frequencies and sequence organization. The reassociation of 300 nucleotide DNA fragments indicates the presence of four classes of DNA differing in repetition frequency. These classes are: highly repetitive sequences, fast intermediate repetitive sequences, slow intermediate repetitive sequences, and unique sequences. The repeated classes are reiterated on average 136,000, 3000, and 42 times respectively. A minor part of the genome is made up of palindromes. — The organization of DNA sequences in the P. sativum genome was determined by the reassociation kinetics of DNA fragments of varying length. Further information was derived from S1 nuclease resistance and from hyperchromicity measurements on DNA fragments reassociated to defined C₀t values. — The portion of the genome organized in a short period interspersion pattern amounts to 47%, with the unique sequences on an average 1000 nucleotides long, and most of the repetitive sequences about 300 nucleotides in length, whereas the weight average length may be up to 600 nucleotides. — About 5% unique DNA and 11% slow intermediate repetitive DNA consist of sequences from 10³ up to 10⁴ nucleotides long; these are interspersed with repetitive sequences of unknown length. Long repetitive sequences constitute 33% of the genome, 13% are satellite-like organized, and 20% in long stretches of intermediate repetitive DNA in which highly divergent sequences alternate with sequences that show only minimal divergence. — The results presented indicate remarkable similarities with the genomes of most animal species on which information is available. The most intriguing pecularity of the plant genome derives from its high content of repetitive DNA and the presumed organization of the latter.     

Correlation approach to identify coding regions in DNA sequences.

Recently, it was observed that noncoding regions of DNA sequences possess long-range power-law correlations, whereas coding regions typically display only short-range correlations. We develop an algorithm based on this finding that enables investigators to perform a statistical analysis on long DNA sequences to locate possible coding regions. The algorithm is particularly successful in predicting the location of lengthy coding regions. For example, for the complete genome of yeast chromosome III (315,344 nucleotides), at least 82% of the predictions correspond to putative coding regions; the algorithm correctly identified all coding regions larger than 3000 nucleotides, 92% of coding regions between 2000 and 3000 nucleotides long, and 79% of coding regions between 1000 and 2000 nucleotides. The predictive ability of this new algorithm supports the claim that there is a fundamental difference in the correlation property between coding and noncoding sequences. This algorithm, which is not species-dependent, can be implemented with other techniques for rapidly and accurately locating relatively long coding regions in genomic sequences.     

A small nuclear precursor of messenger RNA in the cellular slime mold Dictyostelium discoideum

Previous work (Firtel et al., 1972) showed that messenger RNA from the cellular slime mold Dictyostelium discoideum, like that from mammalian cells, contains a sequence of about 100 adenylic acid residues at the 3′ end. We show here that Dictyostelium nuclei, labeled under a variety of conditions, do not contain material analogous to the large nuclear heterogeneous RNA found in mammalian cells. Rather, the majority of pulse-labeled nuclear RNA that is not a precursor of ribosomal RNA does contain at least one sequence of polyadenylic acid; this RNA, with an average molecular weight of 500,000, appears to be only 20% larger than cytoplasmic messenger RNA.Pulse-labeling experiments show that the nuclear poly(A)-containing RNA is a material precursor of messenger RNA. Whereas previous work showed that over 90% of messenger RNA sequences are transcribed from non-reiterated DNA, we show here that about 25% of nuclear poly (A)-containing RNA is transcribed from reiterated DNA sequences and only 75% from single-copy DNA. We present evidence that a large fraction of the nuclear poly(A)-containing RNA contains, at the 5′ end, a sequence of about 300 nucleotides that is transcribed from repetitive DNA, and which is lost before transport of messenger RNA into the cytoplasm.Based on these and other results, we present a model of arrangement of repetitive and single-copy DNA sequences in the Dictyostelium chromosome.     

Direct demonstration of a complementarity between mRNA and double-stranded sequences of pre-mRNA

The total poly(A)-containing mRNA from mouse liver or Ehrlich ascites carcinoma cells was annealed with denatured ds RNA prepared from heavy nuclear ³H-labeled pre-mRNA of the same tissue. The hybrids formed were detected by binding of complexes to poly(U)-Sepharose columns through the poly(A) of mRNA. With this technique, about 30% of labeled ds RNA was bound to poly(U)-Sepharose after annealing it with an mRNA excess. The proportion of hybrid material detected by RNase treatment was two to three times lower than that obtained by poly(U)-Sepharose binding. The length of the RNase-stable acid precipitable hybrid material consisted of heterogeneous sequences of 10–100 nucleotides long when cytoplasmic, and 10–60 nucleotides long when polysomal mRNA was used in the hybridization reaction. The results obtained show that at least some of the mRNA molecules contain sequences complementary to one of the branches of the pre-mRNA hairpins. These results are compatible with the idea that the hairpin-like sequences in pre-mRNA are localized between mRNA and the non-informative part of the precursor molecule.     

Repetitive DNA in Yeasts

BETWEEN 10% and 70% of the nuclear DNA of all higher organisms consists of repeating sequences^1,2 (in some organisms only 6–13 base pairs long³) which comprise families of identical or similar base sequences repeated from several hundred to more than a million times. Much of this is not transcribed⁴ and the most repetitive sequences are located in the centromeric heterochromatin⁵. If repetitive DNA occurs in all eukaryotic cells, however, it is surprising that in renaturation studies it has not been found in yeast^2,6. In Saccharomyces cerevisiae,a large number of the AT base pairs of the mitochondrial DNA probably occur in poly AT sequences^7,8. This may result in unusual renaturation kinetics.     

DNA sequence organization and transcription of the chicken genome

A new approach has been used to examine DNA sequence organization in the chicken genome. The interspersion pattern was determined by studying the fraction of labelled DNA fragments of different lengths that hybridized to an excess of short chicken repeated DNA sequences. The results indicate that chicken DNA has a pattern of sequence organization quite different than the standard ‘Xenopus’ or ‘Drosophila’ patterns. Two classes of unique sequences are found. One, 34% of the genome, consists of unique sequences approx. 4 kb long interspersed with repeated sequences. The second, non-interspersed fraction, 38% of the genome, consists of unique sequences found in long tracts, a minimum of approx. 22 kb in length. In an attempt to determine whether a relationship exists between DNA sequence organization and the distribution of structural genes we have isolated chicken DNA sequences belonging to different interspersion classes and tested each for the presence of structural genes by hybridization to excess poly(A)⁺ mRNA. Sequences complementary to poly(A)⁺ mRNA can be found with approximately the same frequency in both the non-interspersed fraction of the genome and a repeat-contiguous fraction enriched for interspersed sequences.     

Vaccinia virus produces late mRNAs by discontinuous synthesis

We describe the unusual structure of a vaccinia virus late mRNA. In these molecules, the protein-coding sequences of a major late structural polypeptide are preceded by long leader RNAs, which in some cases are thousands of nucleotides long. These sequences map to different regions of the viral genome and in one instance are separated from the late gene by more than 100 kb of DNA. Moreover, the leader sequences map either upstream or downstream of the late gene, are transcribed from either DNA strand, and are fused to the late gene coding sequence via a poly(A) stretch. This demonstrates that vaccinia virus produces late mRNAs by tagging the protein-coding sequences onto the 3' end of other RNAs.     

The presence of polyriboadenylic acid sequences in calf lens messenger RNA

The presence of poly(rA) sequences in lens RNA has been demonstrated by the isolation of RNase A and T₁-resistant fragments of approximately 50 nucleotide residues. These poly(rA)-rich sequences, obtained from lenses incubated for six hours in organ culture with [³H]adenosine, are located at the 3′ termini of mRNA as determined by 3′ exoribonuclease digestion. Limited digestion of the [³H]adenosine-labeled mRNA with the enzyme led to the abolition of binding to poly(rU)-filters and a concomitant loss of template activity with avian myeloblastosis virus RNA-dependent DNA polymerase. Furthermore, after incubation of lenses in organ culture with 3′-deoxyadenosine, the isolated polysomal RNA was unable to function as a template in an avian myeloblastosis virus RNA-dependent DNA polymerase-catalyzed reaction system.     

Contrasting patterns of DNA sequence arrangement in Apis mellifera (Honeybee) and Musca domestica (housefly)

We have examined the organization of the repeated and single copy DNA sequences in the genomes of two insects, the honeybee (Apis mellifera) and the housefly (Musca domestica). Analysis of the reassociation kinetics of honeybee DNA fragments 330 and 2,200 nucleotides long shows that approximately 90% of both size fragments is composed entirely of non-repeated sequences. Thus honeybee DNA contains few or no repeated sequences interspersed with nonrepeated sequences at a distance of less than a few thousand nucleotides. On the other hand, the reassociation kinetics of housefly DNA fragments 250 and 2,000 nucleotides long indicates that less than 15% of the longer fragments are composed entirely of single copy sequences. A large fraction of the housefly DNA therefore contains repeated sequences spaced less than a few thousand nucleotides apart. Reassociated repetitive DNA from the housefly was treated with S1 nuclease and sized on agarose A-50. The S1 resistant sequences have a bimodal distribution of lengths. Thirty-three percent is greater than 1,500 nucleotide pairs, and 67% has an average size about 300 nucleotide pairs. The genome of the housefly appears to have at least 70% of its DNA arranged as short repeats interspersed with single copy sequences in a pattern qualitatively similar to that of most eukaryotic genomes.     

CHARACTERIZATION OF POLY(A) SEQUENCES IN BRAIN RNA

—Nuclear and polysomal brain RNA from the rabbit bind to Millipore filters and oligo(dT)-cellulose suggesting the presence of poly(A) sequences. The residual polynucleotide produced after RNase digestion of ³²P pulse-labelled brain RNA is 95% adenylic acid and 200-250 nucleotides in length. After longer isotope pulses the polysomal poly(A) sequence appears heterodisperse in size and shorter than the nuclear poly (A). Poly(A) sequences of brain RNA are located at the 3′-OH termini as determined by the periodate-[³H]NaBH₄ labelling technique. Cordycepin interferes with the processing of brain mRNA as it inhibits in vivo poly(A) synthesis by about 80% and decreases the appearance of rapidly labelled RNA in polysomes by about 45%. A small poly(A) molecule 10-30 nucleotides in length is present in rapidly labelled RNA. It appears to be less sensitive to cordycepin than the larger poly(A) and is not found in polysomal RNA.