The organization of the main component DNA of a crustacean genome with a paucity of middle repetitive sequences

The frequency classes and organization of the main component (mc) DNA of a crustacean, the land crab, Gecarcinus lateralis, have been characterized. The reassociation kinetics of 380 nucleotide long mcDNA fragments show that approximately 50% contain sequences repeated more than 800 times. Present in few, if any, copies are sequences repeated from 2 to 800 times. The remainder of the DNA reassociates as single copy sequences with a rate constant consistent with the organism's genome size. The reassociation kinetics of highly sheared DNA fragments of every true crab studied (Vaughn, 1975; Christie et al., 1976) are similar to each other and different from those of other invertebrate DNAs (Goldberg et al., 1975). Each of these genomes has a paucity of sequences repeated from 10 to 800 times and an abundance of highly repeated sequences. To determine if sequences repeated more than 800 times are interspersed with single copy sequences, we examined the arrangement of repetitive and non-repetitive sequences in mcDNA. The reassociation and melting properties of partially duplex mcDNA fragments of increasing lengths show that at least 75% of the DNA is organized in an interspersed pattern. In this pattern, single copy sequences with an average length of 800–900 nucleotides are interspersed with repetitive sequences. S1 nuclease digestion of reassociated 3100 nucleotide fragments indicates that 44% of the mcDNA is repetitive and that one-third of the repetitive sequences (average length=285 nucleotides) are interspersed with single copy sequences. We conclude that repetitive sequencies are interspersed with most of the single copy sequences in an interspersion pattern similar to that of Xenopus rahter than to that of another arthropod, Drosophila.Operated by Union Carbide Corporation for the Energy Research and Development Administration     

Specific association of repetitive DNA sequences with major histocompatibility genes.

The DNA sequence organization of a 17.8-kilobase segment of porcine DNA, containing a functional major histocompatibility (MHC) gene, has been studied. The DNA flanking the MHC gene contains at least 10 distinct repetitive DNA sequence elements, each of which occurs only once within the 17.8-kilobase DNA segment. Their reiteration frequencies in the genome range from 10(2) to 10(4). The genomic organization of seven of these sequence elements has been examined; all are interspersed with other, unrelated DNA sequences. These seven repeated sequences are not generally associated in the genome. However, they appear to be nonrandomly linked in MHC-associated regions of the genome: at least two additional DNA segments containing MHC-homologous DNA also contain sequences homologous to DNA fragments bearing the seven different repeats. Of the seven sequences, four can be detected in splenic total RNA. These results suggest that these repeated elements are specifically associated with the MHC locus.     

Genome size and complexity of the obligate fungal pathogen,Bremia lactucae

The size and organization of the genome of Bremia lactucae, a highly specialized fungal pathogen of lettuce, has been characterized using dot blot genomic reconstructions, reverse genomic blots, and genomic DNA reassociation kinetics. The haploid genome contains 5 × 10⁷ bp of DNA and 65% of the nuclear DNA is repeated. Low copy sequences are interspersed with repeated sequences in a short-period interspersion pattern. This pattern of genome organization is different to that described for other fungi. Although most fungi have been shown to contain some form of repetitive DNA other than the ribosomal repeat, the high percentage of repetitive DNA and the interspersion of low copy and repeated sequences are atypical of fungi characterized previously.     

DNA sequence organization in the genome of Nicotiana tabacum

The genome of Nicotiana tabacum was investigated by DNA/DNA reassociation for its spectrum of DNA repetition components and pattern of DNA sequence organization. The reassociation of 300 nucleotide DNA fragments analyzed by hydroxyapatite chromatography reveals the presence of three major classes of DNA differing in reiteration frequency. Each class of DNA was isolated and characterized with respect to kinetic homogeneity and thermal properties on melting. These measurements demonstrate that the genome of N. tabacum has a 1C DNA content of 1.65 pg and that DNA sequences are represented an average of 12,400, 252, and 1 times each. — The organization of the DNA sequences in the N. tabacum genome was determined from the reassociation kinetics of long DNA fragments as well as S1 nuclease resistance and hyperchromicity measurements on DNA fragments after annealing to C₀t values at which only repetitive DNA sequences will reassociate. At least 55% of the total DNA sequences are organized in a short period interspersion pattern consisting of an alternation of single copy sequences, averaging 1400 nucleotides, with short repetitive elements approximately 300 nucleotides in length. Another 25% of the genome contains long repetitive DNA sequences having a minimal genomic length of 1500 nucleotides. These repetitive DNA sequences are much less divergent than the short interspersed DNA sequence elements. These results indicate that the pattern of DNA sequence organization in the tobacco genome bears remarkable similarity to that found in the genomes of most animal species investigated to date.     

Characterization of the nuclear genome of pearl millet.

The nuclear genome of pearl millet has been characterized with respect to its size, buoyant density in CsCl equilibrium density gradients, melting temperature, reassociation kinetics and sequence organization. The genome size is 0.22 pg. The mol percent G + C of the DNA is calculated from the buoyant density and the melting temperature to be 44.9 and 49.7%, respectively. The reassociation kinetics of fragments of DNA 300 nucleotides long reveals three components: a rapidly renaturing fraction composed of highly repeated and/or foldback DNA, middle repetitive DNA and single copy DNA. The single copy DNA consists of 17% of the genome. 80% of the repetitive sequences are at least 5000 nucleotide pairs in length. Thermal denaturation profiles of the repetitive DNA sequences show high Tm values implying a high degree of sequence homogeneity. About half of the single copy DNA is short (750--1400 nucleotide paris) and interspersed with long repetitive DNA sequences. The remainder of the single copy sequences vary in size from 1400 to 8600 nucleotide pairs.     

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.     

Recombination in simian virus 40-infected cells. Structure of naturally arising variants ev-2114, ev-2102, and ev-1110

The complete nucleotide sequence has been determined for three newly cloned evolutionary variants from two different independently generated evolutionary series (1100 and 2100 series) of simian virus 40 (SV40). These naturally arising variants, designated ev-1110, ev-2102, and ev-2114, were isolated after five high multiplicity serial passages. The structure of the variants consists of a monomeric unit tandemly repeated four times (ev-2102 and ev-2114) or six times (ev-1110) in the variant genome; the variants have four or six copies, respectively, of the viral origin signal for DNA replication. The DNA content in the three variants is vastly different in that the genome of variant ev-2114 contains only rearranged viral sequences, while variant ev-2102 contains a substitution with monkey DNA sequences consisting of a nearly complete dimeric unit of Alu family sequences as well as less repetitive sequences and variant ev-1110 contains monkey DNA sequences derived solely from repetitive alpha-component DNA. Recombination events, cellular sequences, and structural features of these and other naturally arising SV40 variants are compared.     

A study of the evolution of repeated DNA sequences in primates and the existence of a new class of repetitive sequences in primates.

A new class of lowly repetitive DNA sequences has been detected in the primate genome. The renaturation rate of this sequence class is practically indistinguishable from the renaturation rate of single-copy sequences. Consequently, this lowly repetitive sequence class has not been previously observed in DNA renaturation rate studies. This new sequence class is significant in that it might occupy a major fraction of the primate genome.Based on a study of the thermal stabilities of DNA heteroduplexes constructed from human DNA and either bonnet monkey or galago DNAs, we are able to compare the relative mutation rates of repetitive and single-copy sequences in the primate genome. We find that the mutation rate of short, interspersed repetitive sequences is either less than or approximately equal to the mutation rate of single-copy sequences. This implies that the base sequence of these repetitive sequences is important to their biological function.We also find that numerous mutations have accumulated in interspersed repeated sequences since the divergence of galago and human. These mutations are only recognizable because they occur at specific sites in the repeated sequence rather than at random sites in the sequence. Although interspersed repetitive sequences from human and galago can readily cross-hybridize, these site-specific mutations identify them as being two distinct classes. In contrast, far fewer site-specific mutations have occurred since the divergence of human and monkey.     

Isolation and characterization of IS elements repeated in the bacterial chromosome

Shigella sonnei contains repetitive sequences, including an insertion element IS1, which can be isolated as double-stranded DNA fragments by DNA denaturation and renaturation and by treatment with S1 nuclease. In this paper, we describe a method of cloning the IS1 fragments prepared by the S1 nuclease digestion technique into phage M13mp8 RFI DNA. Several clones contained IS1, usually with a few additional bases. We isolated and characterized five other repetitive sequences using this method. One sequence, 1264 base-pairs in length, had terminal inverted repeats and contained two open reading frames. This sequence, called IS600, showed about 44% sequence homology with IS3 and was repeated more than 20 times in the Sh. sonnei chromosome. Another sequence (named IS629, 1310 base-pairs in length), which was repeated six times, was found also to be related to IS3 and thus IS600. Two other sequences (named IS630 and IS640, 1159 and 1092 base-pairs in length, respectively), which were repeated approximately ten times, had characteristic terminal inverted repeats and contained a large open reading frame coding for a protein. The inverted repeat sequences of IS630 were similar to the sequence at one end of IS200, a Salmonella-specific IS element. The fifth sequence, repeated ten times in Sh. sonnei, had about 98% sequence homology with a portion of IS2. The method described here can be applied to the isolation of IS or iso-IS elements present in any other bacterial chromosome.     

DNA sequence organization in the genome of Cycas revoluta

The pattern of DNA sequence organization in the genome of Cycas revoluta was analyzed by DNA/DNA reassociation. Reassociation of 400 base pair (bp) fragments to various C₀t values indicates the presence of at least four kinetic classes: the foldback plus very highly repetitive sequences (15%), the fast repeats (24%), the slow repeats (44%), and the single copy (17%). The latter component reassociates with a rate constant 1×10^–4 M^–1S^–1 corresponding to a complexity of 1.6× 10⁶ kb per haploid genome. A haploid C. revoluta nucleus contains approximately 10.3 pg DNA. The single-copy sequences account for about 28% of the DNA, but only 17% reassociate with single-copy kinetics because of interspersion with repetitive sequences. — The interspersion of repetitive and single-copy sequences was examined by reassociation of DNA fragments of varying length to C₀t values of 70 and 500. A major (65%) and homogeneous class of single-copy sequences averaging 1,100 bp in length is interspersed in a short period pattern with repeated sequences. A minor (35%) heterogeneous single-copy component is interspersed in a long-period pattern. The majority of repetitive sequences have a length distribution of 100–350 bp with subclasses averaging 150 and 300 bp in length. Repeat sequences with a wide range in sizes exceeding 2 kilobase pair (kb) are also present in this genome. — The size and distribution of inverted repeat (ir) sequences in the DNA of C. revoluta were studied by electron microscopy. It is estimated that there are approximately 4 × 10⁶ ir pairs (one per 2.33 kb) that form almost equal numbers of looped and unlooped palindromes. This high value is 2.5 times that found in wheat DNA. These palindromes are in general randomly distributed in the genome with an average interpalindrome distance of 1.6 kb. The majority (about 85%) of ir sequences of both types of palindromes belong to a main-size class, with an average length of 210 bp in the unlooped and and 163 bp in the looped type. These values are comparable to those reported for some other plant and animal genomes. Distribution of length of single stranded loops showed a main-size class (75%) with an average length of 220 bp.     

Interspersion of repetitive and nonrepetitive DNA sequences in the Drosophila melanogaster genome

Cot analysis shows that the haploid Drosophila genome contains 12% rapidly reassociating, highly reiterated DNA, 12% middle repetitive DNA with an average reiteration frequency of 70, and 70% single-copy DNA. The distribution of the middle repetitive sequences in the genome has been studied by an examination in the electron microscope of the structures obtained when middle repetitive sequences present on large DNA strands reassociate and by the hydroxyapatite binding methods developed by Davidson et al. (1973). At least one third by weight of the middle repetitive sequences are interspersed in single-copy sequences. These interspersed middle repetitive sequences have a fairly uniform distribution of lengths from less than 0.5 to 13 kb, with a number average value of 5.6 kb. The average distance between middle repetitive sequences is greater than 13 kb. The data do not exclude the possibility that essentially all of the middle repetitive sequences have the interspersion pattern described above; however, it is possible that some of the middle repetitive sequences of Drosophila are clustered in stretches of length much greater than 13 kb. The interspersion pattern of the middle repetitive sequences in Drosophila is quite different from that which occurs in the sea urchin, in Xenopus, in rat, and probably many other higher eucaryotes.     

Structure of DNA near long tandem arrays of alpha satellite DNA at the centromere of human chromosome 7.

The centromeric regions of human chromosomes contain long tracts of tandemly repeated DNA, of which the most extensively characterized is alpha satellite. In a screen for additional centromeric DNA sequences, four phage clones were obtained which contain alpha satellite as well as other sequences not usually found associated with tandemly repeated alpha satellite DNA, including L1 repetitive elements, an Alu element, and a novel AT-rich repeated sequence. The alpha satellite DNA contained within these clones does not demonstrate the higher-order repeat structure typical of tandemly repeated alpha satellite. Two of the clones contain inversions; instead of the usual head-to-tail arrangement of alpha satellite monomers, the direction of the monomers changes partway through each clone. The presence of both inversions was confirmed in human genomic DNA by polymerase chain reaction amplification of the inverted regions. One phage clone contains a junction between alpha satellite DNA and a novel low-copy repeated sequence. The junction between the two types of DNA is abrupt and the junction sequence is characterized by the presence of runs of A's and T's, yielding an overall base composition of 65% AT with local areas > 80% AT. The AT-rich sequence is found in multiple copies on chromosome 7 and homologous sequences are found in (peri)centromeric locations on other human chromosomes, including chromosomes 1, 2, and 16. As such, the AT-rich sequence adjacent to alpha satellite DNA provides a tool for the further study of the DNA from this region of the chromosome. The phage clones examined are located within the same 3.3-Mb SstII restriction fragment on chromosome 7 as the two previously described alpha satellite arrays, D7Z1 and D7Z2. These new clones demonstrate that centromeric repetitive DNA, at least on chromosome 7, may be more heterogeneous in composition and organization than had previously been thought.     

Characterization of repetitive DNA in rye (Secale cereale)

Nuclear DNA of rye (Secale cereale), a plant species with a relatively large genome (i.e., 18 pg diploid), has been characterized by determination of its content in repetitive sequences, buoyant density, and thermal denaturation properties. The reassociation kinetics of rye DNA reveals the presence of 70 to 75% repeated nucleotide sequences which are grouped into highly (Cot 1) and intermediately repetitive (Cot 1–100) fractions. On sedimentation in neutral CsCl gradients, native, high molecular weight DNA forms an almost symmetrical band of density 1.702 g/cm³. The highly repetitive DNA (Cot 1), on the other hand, is separated into two distinct peaks; the minor component has a density of 1.703 g/cm³ corresponding to that of a very rapidly reassociating fraction (Cot 0.01) which comprises 10 to 12% of the rye genome. The latter DNA contains segments which are repeated 6×10⁵ to 6×10⁶ times. The major peak of the Cot 1 fraction shows a density of 1.707 g/cm³ and consists of fragments repeated about 3.7×10⁴ times. The intermediately repetitive DNA is much more heterogeneous than the Cot 1 fraction and has a low degree of repetition of the order of 8.5×10². The melting behavior of the Cot 1 fraction reveals the presence of a high degree of base pairing (i.e., 7% mismatching). When native rye DNA is resolved into fractions differing in GC content by hydroxyapatite thermal column chromatography and these fractions are analyzed for the presence of repetitive sequences, it is observed that the highly redundant DNA (Cot 1) is mostly located in the fraction denaturing between 80° and 90°C. This result suggests that highly repetitive rye DNA occurs in a portion of the genome which is neither very rich in AT nor in GC.     

Organization of repetitive DNA sequences in the genome of the echinoderm Holothuria tubulosa.

The abundance of repetitive DNA in the haploid sea cucumber genome has been determined by screening a Holothuria genomic DNA library for clones containing repeated sequences using reverse genome hybridization. Analysis by in situ plaque hybridization of a set of 1132 clones has revealed the presence of repetitive DNA sequences in about 38.1% of the clones screened. The distribution of the reiterated DNA has been further analyzed by restriction endonuclease digestion of seven randomly selected repetitive clones. The repeated sequences have a fairly uniform distribution of lengths with an average length value of 7.3 kb. Analysis of the measurements suggests that the repetitive sequences are interspersed among longer single copy sequences with an average spacing interval of about 47.3 kb indicating that the repetitive and single copy DNA in the Holothuria genome are arranged in a long-period interspersion pattern.     

HpaII library indicates ‘methylation-free islands’ in wheat and barley

Summary A library of wheat genomic DNA HpaII tiny fragments (HTF), sized below 500 bp, has been constructed. Of the clones in the library 80% belong to the single/low-copy category, while 12% of the clones are nuclear repetitive sequences and 8% originate from the chloroplast and mitochondrial DNA. This result shows a substantial enrichment in the single/low-copy sequences of the wheat genome, which contains at least 80% repetitive sequences. Twenty-nine random single/lowcopy clones were analysed further for wheat chromosome location, cross-hybridisation to barley DNA and their association with rare-cutting, C-methylation-sensitive restriction sites. The results show that the HTF clones are associated more frequently than expected with NotI, MluI, NruI and PstI sites in wheat and barley genomic DNA. The 12% repetitive fraction of the clones contain both moderately and highly repetitive sequences, but no tandemly repeated sequences. The level of enrichment for single/low-copy sequences indicates that libraries of this type are a valuable source of probes for RFLP mapping. In addition, the close association of the HTF clones with rare-cutting restriction enzyme sites ensures that HTF clones will have a useful role in the construction of long-range physical maps in wheat.     

Sequence organization of the human genome

The organization of three sequence classes—single copy, repetitive, and inverted repeated sequences—within the human genome has been studied by renaturation techniques, hydroxylapatite binding methods, and DNA hyperchromism. Repetitive sequence classes are distributed throughout 80% or more of the genome. Slightly more than half of the genome consists of short single copy sequences, with a length of about 2 kb interspersed with repetitive sequences. The average length of the repetitive sequences is also small and approximates the length of these sequences found in other organisms. The sequence organization of the human genome therefore resembles the sequence organization found in Xenopus and sea urchin. The inverted repeats are essentially randomly positioned with respect to both sequence class and sequence arrangement, so that all three sequence classes are found to be mutually interspersed in a portion of the genome.     

S1 nuclease definition of highly repeated DNA sequences in the Guinea pig, Cavia porcellus.

Native DNA of the Guinea pig, Cavia porcellus, purified from liver or tissue culture cells, was heat denatured and reassociated to a Cot value of 0.01 (equivalent Cot value of 7.2 x 10(-2)). The reassociated DNA was isolated by digestion with the single-strand DNA specific enzyme S1 nuclease. Spectrophotometric and radioactivity assays demonstrated that 24% of the total DNA was resistant to S1 nuclease treatment. Zero-time reassociation indicated that approximately 3% of the DNA was inverted repeat sequences. Thus, highly repeated sequences comprised 21% of the total genome. CsCl buoyant density ultracentrifugation indicated that this fraction was composed of both main band and satellite sequences. Although actinomycin D - CsCl density gradients failed to give significant separation of the repetitive sequences, distamycin A - CsCl gradients were able to fractionate the DNA into several overlapping bands. The heterogeneity of the repetitive DNA was further demonstrated by the first derivative plots calculated from their thermal denaturation profiles. This analysis revealed six major thermalytes which indicate that there may be at least six discrete components in the repetitive DNA.     

The genome of Zea mays,its organization and homology to related grasses

The pattern of genome organization of Zea mays has been analyzed, and the relationship of maize to possible progenitor species assessed by DNADNA hybridization. Reassociation of 470 and 1,350 bp fragments of maize DNA to various C₀t values demonstrates that the genome is composed of 3 major kinetic classes: highly repetitive, mid-repetitive, and unique. Mini-C₀t curves of the repetitive sequences at short fragment length indicate that the highly repetitive sequence class is 20% of the genome and is present at an average reiteration frequency of 800,000 copies; the mid-repetitive sequence class is 40% of the genome and is present at an average reiteration frequency of 1,000 copies. Thermal denaturation studies show that the highly repetitive sequences are 12% divergent and mid-repetitive sequences are 6% divergent. Most of the genome is organized in two interspersion patterns. One, approximately one-third of the genome, is composed of unique sequences of average length 2,100 bp interspersed with mid-repetitive sequences; the other, also one-third of the genome, is mid-repetitive sequences interspersed with highly repetitive sequences. The repetitive sequences are 500 to 1,000 bp by electron microscopic measurement. The remaining third of the genome is unique sequences farther than 5,000 bp from a palindromic or repetitive sequence. Hybridization of maize DNA from Midwestern Dent to popcorn and related grasses indicates that both the unique and repetitive sequence elements have diverged. Teosinte and popcorn are approximately equally divergent from Midwestern Dent whereas Tripsacum is much more divergent. The divergence times calculated from the depression of T_m in heterologous duplexes indicate that the divergence within Zea mays and between maize and near relatives is at least an order of magnitude greater than expected. This high degree of divergence may reflect the pressures of domestication of maize.