Restriction enzyme and fluorochrome banding analysis of the constitutive heterochromatin of Saguinus species (Callitrichidae, Primates)

Metaphases of Saguinas fuscicollis fuscicollis and Saguinas mystax were subjected to restriction enzyme banding (Alu I, Hae III, Hin fI, Rsa I, Dde I, Mbo I and Msp I) and sequenced C-banding, together with fluorochrome staining (CMA3 and DAPI). Both species showed large C-bands in the pericentromeric regions. S. f. fuscicollis also manifested distal C-bands in both arms of pair 5 and in the short arms of pairs 8-15. In each species the heterochromatin revealed different reactions to the restriction enzymes and fluorochromes. This was related to its location in the genome (centromeric, pericentromeric, distal), making possible the identification of distinct categories of constitutive heterochromatin. In S. f. fuscicollis there were at least five types, namely centromeric in bi-armed chromosomes, centromeric in acrocentrics, pericentromeric, distal, and cryptic bands, detected only with the Alu I. There were three types in S. mystax, viz centromeric in bi-armed chromosomes, centromeric in acrocentric, and pericentromeric chromosomes. Several aspects of their constitution and origin are discussed.     

Characterization of constitutive heterochromatin of Callithrix geoffroyi (Callitrichidae, Primates) by restriction enzymes and fluorochrome bands

The neotropical primate genus Callithrix comprises two groups of species, jacchus and argentata, which inhabit distinct geographical regions and manifest different fur coloration and constitutive heterochromatin (CH) markers in their karyotypes. In this investigation the CH of a representative of the jacchus group, Callithrix geoffroyi, was analysed using fluorochromes and restriction enzymes in situ. To clarify the source of the constitutive heterochromatin of both groups, the data obtained in the jacchus group were compared with those published in the argentata group obtained by the same techniques. The C-bands of C. geoffroyi (four specimens, 2n = 46) were centromeric in all chromosomes, and distally located in pairs 6 and 22. The Alu I, Hae III, Hin fI, Rsa I, Dde I, Mbo I, and Msp I restriction endonucleases and CMA3 and DAPI fluorochromes produced different bands, which allowed the characterization of four distinct types of constitutive heterochromatin in the C. geoffroyi genome. Several of these types of heterochromatin were present in the ancestor of the two groups of species, jacchus and argentata, while others originated after their cladogenesis.     

A comparative study on proteins released from metaphase chromosomes after digestion with restriction endonucleases and deoxyribonuclease I

Extensive digestion of Chinese hamster metaphase chromosomes with Alu I, Hae III and Hinf I released up to 40 distinct chromosomal proteins. Some of the proteins released by Hae III or Hinf I were enriched in the protein moiety liberated by Alu I but several proteins released by Hae III were not released by Alu I digestion. The amount of chromosomal protein released by deoxyribonuclease I (DNase I) was comparable to that liberated by the three restriction enzymes so far tested, while only four abundant protein species were detectable in the protein moiety released by DNase I. Two of them with molecular weights of 58,000 and 50,000 were also released by the three restriction enzymes and are similar in size to those found previously in the core-like structure of histone-depleted chromosomes.     

伊氏锥虫动基体DNA微环的研究

采用限制性内切酶消化、琼脂糖凝胶电泳及分子杂交技术对８株中国伊氏锥虫动基体ＤＮＡ微环进行了比较研究。结果显示,我国伊氏锥虫株之间的ｋＤＮＡ微环序列具有较高的同源性,仅限制酶ＡｌｕＩ,ＨｉｎｆＩ及ＭｂｌＩ的酶解结果显示少数虫株的ｋＤＮＡ微环存在异源序列。这种异源性可以作为伊氏锥虫种内分类的遗传学标志。     

The pattern of restriction enzyme-induced banding in the chromosomes of chimpanzee,gorilla, and orangutan and its evolutionary significance

Summary The pattern of banding induced by five restriction enzymes in the chromosome complement of chimpanzee, gorilla, and orangutan is described and compared with that of humans. The G banding pattern induced by Hae III was the only feature common to the four species. Although hominid species show almost complete chromosomal homology, the restriction enzyme C banding pattern differed among the species studied. Hinf I did not induce banding in chimpanzee chromosomes, and Rsa I did not elicit banding in chimpanzee and orangutan chromosomes. Equivalent amounts of similar satellite DNA fractions located in homologous chromosomes from different species or in nonhomologous chromosomes from the same species showed different banding patterns with identical restriction enzymes. The great variability in frequency of restriction sites observed between homologous chromosome regions may have resulted from the divergence of primordial sequences changing the frequency of restriction sites for each species and for each chromosomal pair. A total of 30 patterns of banding were found informative for analysis of the hominid geneaalogical tree. Using the principle of maximum parsimony, our data support a branching order in which the chimpanzee is more closely related to the gorilla than to the human.     

Tandem repeats within the inverted terminal repetition of vaccinia virus DNA

A tandemly repeated sequence within the genome of vaccinia virus is cut to fragments of approximately 70 bp by Hinf I, Taq I or Mbo II. The 70 bp repetition was localized within the much larger (10,300 bp) inverted terminal repetition by restriction analysis of cloned DNA fragments and by hybridization of the purified 70 bp repeat to vaccinia virus DNA restriction fragments. The molar abundance of the 70 bp fragment corresponds to a 30 fold repetition at each end of the genome. The repeating restriction endonuclease sites were mapped by agarose gel electrophoresis of partial Hinf I digests of the terminally labeled cloned DNA fragment. The first of 13 repetitive Hinf I sites occurred approximately 150 bp from the end of the cloned DNA. After an intervening sequence of approximately 435 bp, a second series of 17 repetitive Hinf I sites occurred. The DNA between the two blocks of repetitions has a unique sequence containing single Dde I, Alu I and Sau 3A sites. Tandem repeats within the inverted terminal repetition could serve to accelerate self-annealing of single strands of DNA to form circular structures during replication.     

The banding pattern produced by restriction endonucleases in mouse chromosomes

The metaphase chromosomes of mouse have been treated with the restriction endonucleases Alu I, Mbo I, Hae III and Eco RII and subsequently stained with the DNA-specific dye Ethidium bromide. A striking correspondence between previous biochemical findings and our cytological results has been noticed with Alu I, Mbo I and Hae III, which were capable of digesting all but the DNA localized at the centric constitutive heterochromatic areas. Digestion with Eco RII, on the contrary, resulted in cytological data which apparently did not fit in with the biochemical results previously obtained by digesting the DNA of this species with the same enzyme. It is postulated that factors such as chromatin organization, in addition to DNA base sequence, can determine the results we report.     

Genomic variability within laboratory and wild isolates of the trichostrongyle mouse nematode Heligmosomoides polygyrus

PCR-RFLP techniques have been used to characterize wild and laboratory isolates of the trichostrongyle nematode Heligmosomoides polygyrus from the wood mouse Apodemus sylvaticus and the laboratory mouse Mus musculus respectively. Both isolates can be distinguished by eight endonuclease digestions of the ITS region of the rDNA repeat namely, Alu I, Dde I, Hpa II, Hae III, Hinf I, Hha I, Pvu II and Sal I. In two of the digests, Hinf I and Rsa I, a minor polymorphism was observed in the wild isolate of H. polygyrus which has been cultured in laboratory-bred A. sylvaticus for several generations when compared with H. p. polygyrus from wild A. sylvaticus. A minor polymorphism was also identified in further wild isolates of H. polygyrus collected from A. sylvaticus in a field site in Egham, Surrey. However no evidence of polymorphism was observed in the laboratory isolate of H. polygyrus from the CD1 strain of M. musculus and the laboratory-bred A. sylvaticus. Reasons for this are discussed and further studies on the population genetics of H. polygyrus are suggested.     

Restriction enzyme banding of mouse metaphase chromosomes

Fixed metaphase chromosomes from mouse strain RIII embryos or A9 cells were treated with a restriction endonuclease, followed by Giemsa staining. Aha I, Hinf I, or Mbo I treatment produced a C-band pattern, and Eco RII or Hae III produced a G-band plus C-band pattern. Ava II and Bst NI each produced a G-band pattern, but on most chromosomes only a small segment of each C-band, adjacent to the centromere, was stained. These tiny residual C-bands may contain a minor satellite located adjacent to the major satellite clusters.     

Cleavage map of bacteriophage phiX174 RF DNA by restriction enzymes.

phiX RF DNA was cleaved by restriction enzymes from Haemophilus influenzae Rf (Hinf I) and Haemophilus haemolyticus (Hha. I). Twenty one fragments of approximately 25 to 730 base pairs were produced by Hinf I and seventeen fragments of approximately 40 to 1560 base pairs by Hha I. The order of these fragments has been established by digestion on Haemophilus awgyptius (Hae III) and Arthrobacter luteus (Alu I) endonuclease fragments of phiX RF with Hinf I and Hha1. By this method of reciprocal digestion a detailed cleavage map of phiX RF DNA was constructed, which includes also the previously determined Hind II, Hae III and Alu I cleavage maps of phiX 174 RF DNA (1, 2). Moreover, 28 conditional lethal mutants of bacteriophage phiX174 were placed in this map using the genetic fragment assay (3).     

Prometaphase chromosomes of the howler monkey (Aloutta caraya): G,C, NOR,and restriction enzyme (RES) banding

Prometaphase lymphocyte chromosomes from eight adult argentinian Alouatta caraya females were characterized using sequential G-C banding techniques, Ag-NOR bands and bands obtained with the restriction enzymes Hae III, Eco RI, Alu I and Sau 3A. The cytogenetic analysis showed 2n = 52, with four, five, or six NOR chromosomes. Digestion with Hae III and Eco RI produced G-like-bands. Centromere regions and two interstitial C-bands (in chromosomes number 16 and 21) showed intraindividual or interindividual heterochromatic polymorphisms. Alu I digestion produced C-like bands with gaps in the centromere regions, and Sau 3A produced C-like bands. The karyotypes and banding patterns of A. caraya, A. palliata, A. belzebul, and A. seniculus are compared, based on whole chromosome and whole arm homeologies. © 1994 Wiley-Liss, Inc.     

The karyotype of the South American rodent Kunsia tomentosus (Lichtenstein, 1830).

Chromosomal analysis of Kunsia tomentosus showed a karyotype with 2n = 44, constituted by 21 pairs of acrocentric autosomes. The X chromosome was a median acrocentric, between pairs 3 and 4 in size, and the Y chromosome was a small acrocentric (between pairs 19 and 20). Five pairs with nucleolus organizer regions were located at the short arms. C-banding showed blocks of constitutive heterochromatin occurring in the centromeres of all autosomes and of the X chromosome. The Y chromosome was entirely heterochromatic. In order to identify possible homologies, karyotypes of Kunsia and Scapteromys, the phyletically related taxa, were compared. No autosome shared by either genus was found by G-band comparisons. The C-band patterns and those produced by Alu I, Mbo I, Rsa I and Hae III restriction endonucleases were also different. The results of FISH indicated a different composition of the telomeric regions of the chromosomes of both taxa, since in Scapteromys the probes hybridized in both telomeres, and in Kunsia this hybridization only occurred in one of the telomeres. These differences also occurred in the localization and number of nucleolus organizer regions.     

Genetic variation in native and introduced populations of the 'New Zealand flatworm', Arthurdendyus triangulatus

The internal transcribed spacer regions (ITS1 and ITS2) including the 5.8S region of the ‘New Zealand flatworm’, Arthurdendyus triangulates, are 1004 base pairs in length. Restriction fragment length polymorphism analysis of PCR products (PCR‐RFLP) was conducted on A. triangulates specimens from 45 locations in Northern Ireland, Scotland, England and New Zealand. Seven restriction endonucleases (Alu I, Rsa I, Sau3A I, Cfo I, Nde I, Dde I, and Mbo I) were used to reveal intraspecific variation. Analysis of molecular variance revealed the presence of population genetic substructuring, with most genetic heterogeneity present between populations rather than between individuals or geographical regions. No distinct differences were found between Northern Irish and Scottish populations but phylogenetic analysis supports the hypothesis of multiple introductions from New Zealand. There was no significant relationship between genetic distance and geographic distance, as would be expected for natural spread, indicating that this species is largely anthropochorous, even in parts of New Zealand.     

The selective digestion of polytene and mitotic chromosomes of Drosophila melanogaster by the Alu I and Hae III restriction endonucleases

Polytene and mitotic chromosomes of Drosophila melanogaster were treated with either Alu I or Hae III restriction endonucleases. Subsequent staining with the DNA-specific fluorochrome ethidium bromide showed that these enzymes are capable of selectively digesting chromosomal DNA in fixed cytological preparations, as previously shown in mammalian metaphase chromosomes. Alu I or Hae III digestion made possible the localization in situ of some highly repetitive DNAs in both polytene and mitotic chromosomes, while only Alu I permitted the localization of the 5S RNA genes on the polytene chromosomes of D. melanogaster.     

A stretch of "late" SV40 viral DNA about 400 bp long which includes the origin of replication is specifically exposed in SV40 minichromosomes.

Examination of DNA fragments produced from either formaldehyde-fixed or unfixed SV40 minichromosomes by multiple-cut restriction endonucleases has led to the following major results: Exhaustive digestion of unfixed minichromosomes with Hae III generated all ten major limit-digest DNA fragments as well as partial cleavage products. In striking contrast to this result, Hae III acted on formaldehyde-fixed minichromosomes to yield only one of the limit-digest fragments, F, which is located in the immediate vicinity of the origin of replication, spanning nucleotides 5169 and 250 on the DNA sequence map of Reddy et al. (1978). This 300 base pair (bp) fragment was released as naked DNA from formaldehyde-fixed, Hae III-digested minichromosomes following treatment either by pronase-SDS or by SDS alone. In the latter case, the remainder of the minichromosome retained its compact configuration as assayed by both sedimentational and electrophoretic methods. In minichromosomes, the F fragment is therefore not only accessible to Hae III at its ends, but is also neither formaldehyde cross-linked into any SDS-resistant nucleoprotein structure nor topologically "locked" within the compact minichromosomal particle. This same fragment was preferentially produced during the early stages of digestion of unfixed minichromosomes with Hae III, and its final yield in the exhaustive Hae III digest was significantly higher than that of other limit-digest fragments. Similar results were obtained upon digestion of either unfixed or formaldehyde-fixed minichromosomes with Alu I. In particular, of approximately twenty major limit-digest DNA fragments, only two fragments (F and P, encompassing nucleotides 5146 to 190, and 190 to 325, respectively) were produced by Alu I from the formaldehyde-fixed minichromosomes. All other restriction endonucleases tested (Mbo I, Mbo II, Hind III, Hin II+III and Hinf I), for which there are no closely spaced recognition sequences in the above mentioned regions of the SV40 genome, did not produce any significant amount of limit-digest DNA fragments from formaldehyde-fixed minichromosomes. These findings, taken together with our earlier data on the preferential exposure of the origin of replication in SV40 minichromosomes (Varshavsky, Sundin and Bohn, 1978), strongly suggest that a specific region of the "late" SV40 DNA approximately 400 bp long is uniquely exposed in the compact minichromosome. It is of interest that, in addition to the origin of replication, this region contains binding sites for T antigen (Tjian, 1977), specific tandem repeated sequences and apparently also the promoters for synthesis of late SV40 mRNAs (Fiers et al., 1978; Reddy et al., 1978).     

Cloning and characterization of a complex satellite DNA from Drosophila melanogaster.

The sequence organization of the 1.688 satellite DNA (density 1.688 g/cm³ in CsCl) has been investigated, and this satellite has been found to differ from the other D. melanogaster satellite DNAs in having a much greater sequence complexity. Purification of 1.688 satellite DNA by successive equilibrium density centrifugations yielded a fraction 77% pure. Segments of satellite DNA were isolated by molecular cloning in the plasmid vector pSC101. One recombinant plasmid contained a segment of 1.688 satellite DNA 5.8 kilobase pairs in size and was stable during propagation in E. coli. Recognition sites for restriction enzymes from Haemophilus aegyptius (Hae III), Haemophilus influenzae f (Hinf) and Arthrobacter luteus (Alu I) were mapped in the satellite DNA of this hybrid plasmid. The spacing of Hae III, Hinf and two Alu I sites at regular intervals of about 365 base pairs is strong evidence that the sequence complexity of this satellite DNA is 365 base pairs. Further evidence comes from the finding that both gradient-purified and cloned 1.688 satellite DNA renature with their Hae III sites in register. The Hae III and Hinf sites in gradient-purified satellite DNA have been shown by Manteuil, Hamer and Thomas (1975) and Shen, Wiesehahn and Hearst (1976) to be distributed at intervals of 365 base pairs and integral multiples thereof. These investigators proposed that some of the sites in an otherwise regular array have been randomly inactivated. Cloned satellite DNA provided a hybridization probe for sensitive studies of the arrangement of these recognition sites in gradient-purified satellite DNA. Some regions of satellite DNA were found to contain many fewer recognition sites than expected from the proposed models. These findings suggest that different regions of 1.688 satellite DNA may exhibit different arrangements of Hae III and Hinf recognition sites.     

A possible structure for calf satellite DNA I.

Calf satellite DNA I (p = 1.715) has been hydrolysed by a number or restriction endonucleases. It consists of a repeating unit of 1460 nucleotide pairs within which the sites of Eco R II Mbo I, Sac I, Alu I, Ava II and Hha I were localised in comparison with those of Eco R I and Hind II. The distribution of the Hpa II, Sac I, Hha I, Hinf I and Mbo II sites within calf satellite DNA I, as well as that of several restriction endonuclease sites within calf satellite DNA III (p = 1.705) allowed me to define subsatellite fractions. Furthermore, some of the sites of the CpG containing restriction enzymes Hpa II and Hha I are lacking. The possible implications of these results are discussed.     

Restriction endonuclease digestion of meiotic chromosomes of Allium subvillosum L.

Meiotic chromosomes of the liliaceous plant Allium subvillosum were characterized by means of digestion with the restriction endonucleases (REs) Hae III and Msp I followed in some cases by treatments with proteinase K or nuclease S1. Both REs are capable of digesting euchromatin, giving a C-like banding pattern. Something similar can be observed when chromosomes are digested with the two restriction endonucleases followed by treatments with proteinase K. By contrast, heterochromatic regions can be digested only after sequential treatments using Hae III plus nuclease S1. These results are discussed in relation to the structural organization of plant meiotic chromosomes as well as the special conformation of plant heterochromatin.     

Relationships Among Group IV Phytophthora Species Inferred by Restriction Analysis of the ITS2 Region

The polymerase chain reaction (PCR) was used to amplify the ITS2 region of nuclear ribosomal DNA from six Phytophthora species which comprise taxonomic Group IV. Digestion of the ca. 600 bp PCR product with restriction enzymes Alu I, Dra I, Hha I, Hinf I, Msp I, and Taq I revealed variation which allowed relationships among the species to be assessed. P. infestans , P. mirabilis and P. phaseoli were indistinguishable from one another with all enzymes tested. With Alu I and Taq I. P. ilicis , P. colocasiae . and P. hibernalis each showed unique banding patterns different from the common banding pattern shared by P. infestans . P. mirabilis . and P. hibernalis . Dra I allowed differentiation of P. ilicis and P. colocasiae from P. infestans , P. mirabilis , P. phaseoli , and P. hibernalis . all of which shared a common banding pattern. Hha I allowed differentiation of P. colocasiae and P. hibernalis from P. infestans, P. mirabilis, P. phaseoli , and P. ilicis . Hinf I allowed differentiation of P. ilicis and P. hibernalis , (each of which showed a unique banding pattern) from P. infestans, P. mirabilis, P. phaseoli , and P. colocasiae . Msp I allowed differentiation of P. hibernalis from the other five species. Species groupings determined by restriction analysis of ITS2 were consistent with those based on morphological criteria. These results show that restriction analysis of PCR-amplified TS2 regions can be useful as an adjunct to morphological criteria in Phytophthora species identification.     

Molecular taxonomy and phylogeny of entomopathogenic nematode species (Rhabditida: Steinernematidae) by RFLP analysis of the ITS region of the ribosomal DNA repeat unit

The ITS region of 19 isolates of the genus Steinernema Travassos, 1927 belonging to 17 species was PCR amplified. The resulting products were then digested with 17 different restriction endonucleases and the fragments generated were separated by agarose gel electrophoresis. Some enzymes yielded patterns which were highly diagnostic (e.g. Alu I, Dde I, Hha I and Hinf I) while others showed similar RFLP patterns for the majority of species (e.g. Hpa II). All of the species could be unequivocally identified using this method. A tree was constructed based on band sharing which resulted in clusters of species which exhibited similar morphological features.