The mechanism and pattern of banding induced by restriction endonucleases in human chromosomes

The mechanism of chromosome banding induced by restriction endonucleases was analyzed by measuring the amount of radioactivity extracted from [¹⁴C]thymidine-labeled chromosomes digested first with restriction enzymes and subsequently with proteinase K and DNase I. Restriction enzymes with a high frequency of recognition sites in the DNA produced a large number of short DNA fragments, which were extracted from chromosomes during incubation with the enzyme. This loss of DNA resulted in decreased chromosomal staining, which did not occur in regions resistant to restriction enzyme digestion and thus led to banding. Subsequent digestion of chromosomes with proteinase K produced a further loss of DNA, which probably corresponded to long fragments retained in the chromosome by the proteins of fixed chromatin. Restriction enzymes induce chromatin digestion and banding in G1 and metaphase chromosomes, and they induce digestion and the appearance of chromocenters in interphase nuclei. This suggests that the spatial organization and folding of the chromatin fibril plays little or no role in the mechanism of chromosome banding.It was confirmed that the pattern of chromosome banding induced by AluI, MboI, HaeIII, DdeI, RsaI, and HinfI is characteristic for each endonuclease. Moreover, several restriction banding polymorphisms that were not found by conventional C-banding were detected, indicating that there may be a range of variability in the frequency and distribution of restriction sites in homologous chromosome regions.     

Improving Illumina assemblies with Hi‐C and long reads: An example with the North African dromedary

Researchers have assembled thousands of eukaryotic genomes using Illumina reads, but traditional mate‐pair libraries cannot span all repetitive elements, resulting in highly fragmented assemblies. However, both chromosome conformation capture techniques, such as Hi‐C and Dovetail Genomics Chicago libraries and long‐read sequencing, such as Pacific Biosciences and Oxford Nanopore, help span and resolve repetitive regions and therefore improve genome assemblies. One important livestock species of arid regions that does not have a high‐quality contiguous reference genome is the dromedary (Camelus dromedarius). Draft genomes exist but are highly fragmented, and a high‐quality reference genome is needed to understand adaptation to desert environments and artificial selection during domestication. Dromedaries are among the last livestock species to have been domesticated, and together with wild and domestic Bactrian camels, they are the only representatives of the Camelini tribe, which highlights their evolutionary significance. Here we describe our efforts to improve the North African dromedary genome. We used Chicago and Hi‐C sequencing libraries from Dovetail Genomics to resolve the order of previously assembled contigs, producing almost chromosome‐level scaffolds. Remaining gaps were filled with Pacific Biosciences long reads, and then scaffolds were comparatively mapped to chromosomes. Long reads added 99.32 Mbp to the total length of the new assembly. Dovetail Chicago and Hi‐C libraries increased the longest scaffold over 12‐fold, from 9.71 Mbp to 124.99 Mbp and the scaffold N50 over 50‐fold, from 1.48 Mbp to 75.02 Mbp. We demonstrate that Illumina de novo assemblies can be substantially upgraded by combining chromosome conformation capture and long‐read sequencing.     

Detection and physical map of a omega tox+-related defective prophage in Corynebacterium diphtheriae Belfanti 1030(-)tox-.

A library of chromosomal DNA from Corynebacterium diphtheriae Belfanti 1030(-)tox- was cloned in the lambda phage vector EMBL4 and screened for sequences homologous to corynephage omega tox+ and the attB1-attB2 region of the C7(-)tox- chromosome. Two portions of the 1030(-)tox- chromosome, 35 and 30.5 kilobases long which contain, respectively, the entire region homologous to corynephage omega tox+ and the attB1-attB2 sites, were mapped with the restriction endonucleases BamHI and EcoRI. Chromosomal DNA from 1030(-)tox- was shown to contain a 15.5-kilobase region that was homologous to ca. 42% of the corynephage omega tox+ genome. These sequences were found to hybridize to three regions of the phage genome and do not contain either the diphtheria tox operon or the attP site. These sequences are distant from the chromosomal region that contains the attB1-attB2 sites. Moreover, unlike other known defective prophages, the physical map of this prophage starts at the cos site and is colinear with the vegetative phage map. The 30.5-kilobase region of the 1030(-)tox- chromosome, which contains the attB1-attB2 sites, has a central core region that is almost identical to the corresponding region of the C7(-)tox- chromosome; however, the flanking sequences in these two strains of C. diphtheriae are different.     

Molecular sexing eutherian mammals

Mammals can be molecular sexed by polymerase chain reaction (PCR) amplification of Y chromosome fragments or coamplification of homologous fragments from both sex chromosomes, which are discriminated by size polymorphism or Y‐specific restriction digestion. Although coamplification of X and Y fragments is more reliable, size polymorphism in homologous fragments is uncommon and Y‐specific restriction site identification requires screening with a battery of enzymes or cloning. Here we describe a simple approach, using ‘double peaks’ in the chromatogram upon direct sequencing of PCR products from males, to identify Y‐specific restriction sites, and demonstrate its utility by application to a range of taxa.     

Ribonucleotide reductase M2 subunit sequences mapped to four different chromosomal sites in humans and mice: functional locus identified by its amplification in hydroxyurea-resistant cell lines

The sites of sequences homologous to a murine cDNA for ribonucleotide reductase (RR) subunit M2 were determined on human and murine chromosomes by Southern blot analysis of interspecies somatic cell hybrid lines and by in situ hybridization. In the human genome, four chromosomal sites carrying RRM2-related sequences were identified at 1p31----p33, 1q21----q23, 2p24----p25, and Xp11----p21. In the mouse, M2 sequences were found on chromosomes 4, 7, 12, and 13 by somatic cell hybrid studies. By Southern analysis of human hydroxyurea-resistant cells that overproduce M2 because of gene amplification, we have identified the amplified restriction fragments as those that map to chromosome 2. To further confirm the site of the functional RRM2 locus, two other cDNA clones, p5-8 and S7 (coding for ornithine decarboxylase; ODC), which are coamplified with RRM2 sequences in human and rodent hydroxyurea-resistant cell lines, were mapped by Southern and in situ hybridization. Their chromosomal map positions coincided with the region of human chromosome 2 (p24----p25) that also contains one of the four RRM2-like sequences. Since this RRM2 sequence and p5-8 and ODC are most likely part of the same amplification unit, the RRM2 structural gene can be assigned to human chromosome 2p24----p25. This region is homologous to a region of mouse chromosome 12 that also carries one of numerous ODC-like sequences. In an RRM2-overproducing mouse cell line, we found amplification of the chromosome 12-specific restriction fragments. Thus, we conclude that mouse chromosome 12 carries the functional locus for RRM2.     

Novel (CA)n marker DXYS241 on the nonrecombinant part of the human Y chromosome

The origin of modern humans can be traced by comparing polymorphic sites in either mitochondria or genomic sequences between humans and other primates. The human Y chromosome has both a non-recombining region and X-Y homologous pseudo-autosomal regions. In the nonrecombining region events during evolution can be directly detected. At least a part of homology between Xq21 and Yp11 is a result of rather recent translocations from the X chromosome to the Y chromosome. DNA markers residing in the nonrecombining region of the human Y chromosome are potentially useful in tracing male-specific gene flow in human evolution. However, the number of available markers in the region is limited. Here, we report a novel X-Y homologous (CA)n repeat locus in the nonrecombining region of the Y chromosome. This marker, DXYS241, has several interesting features. Y- and X-chromosome alleles are distinguishable because the Y-chromosome alleles are shorter than the X-chromosome alleles most of the time. We developed 2 primer sets for specific examination of Y- and X-chromosome alleles. The marker should be useful in establishing relationships between populations based on patrilineal gene flow. Sequences homologous to DXYS241 are also found on the X chromosome of primates. Four events during primate evolution that led to the modern human Y chromosome were identified.     

Ionizing radiation-induced instant pairing of heterochromatin of homologous chromosomes in human cells

Using fluorescence in situ hybridization with human band-specific DNA probes we examined the effect of ionizing radiation on the intra-nuclear localization of the heterochromatic region 9q12-->q13 and the euchromatic region 8p11.2 of similar sized chromosomes 9 and 8 respectively in confluent (G1) primary human fibroblasts. Microscopic analysis of the interphase nuclei revealed colocalization of the homologous heterochromatic regions from chromosome 9 in a proportion of cells directly after exposure to 4 Gy X-rays. The percentage of cells with paired chromosomes 9 gradually decreased to control levels during a period of one hour. No significant changes in localization were observed for chromosome 8. Using 2-D image analysis, radial and inter-homologue distances were measured for both chromosome bands. In unexposed cells, a random distribution of the chromosomes over the interphase nucleus was found. Directly after irradiation, the average inter-homologue distance decreased for chromosome 9 without alterations in radial distribution. The percentage of cells with inter-homologue distance <3 micro m increased from 11% in control cells to 25% in irradiated cells. In contrast, irradiation did not result in significant changes in the inter-homologue distance for chromosome 8. Colocalization of the heterochromatic regions of homologous chromosomes 9 was not observed in cells irradiated on ice. This observation, together with the time dependency of the colocalization, suggests an underlying active cellular process. The biological relevance of the observed homologous pairing remains unclear. It might be related to a homology dependent repair process of ionizing radiation induced DNA damage that is specific for heterochromatin. However, also other more general cellular responses to radiation-induced stress or change in chromatin organization might be responsible for the observed pairing of heterochromatic regions.     

Two Types of Sites Required for Meiotic Chromosome Pairing in Caenorhabditis Elegans

Previous studies have shown that isolated portions of Caenorhabditis elegans chromosomes are not equally capable of meiotic exchange. These results led to the proposal that a homolog recognition region (HRR), defined as the region containing those sequences enabling homologous chromosomes to pair and recombine, is localized near one end of each chromosome. Using translocations and duplications we have localized the chromosome I HRR to the right end. Whereas the other half of chromosome I did not confer any ability for homologs to pair and recombine, deficiencies in this region dominantly suppressed recombination to the middle of the chromosome. These deletions may have disrupted pairing mechanisms that are secondary to and require an HRR. Thus, the processes of pairing and recombination appear to utilize at least two chromosomal elements, the HRR and other pairing sites. For example, terminal sequences from other chromosomes increase the ability of free duplications to recombine with their normal homologs, suggesting that telomere-associated sequences, homologous or nonhomologous, play a role in facilitating meiotic exchange. Recombination can also initiate at internal sites separated from the HRR by chromosome rearrangement, such as deletions of the unc-54 region of chromosome I. When crossing over was suppressed in a region of chromosome I, compensatory increases were observed in other regions. Thus, the presence of the HRR enabled recombination to occur but did not determine the distribution of the crossover events. It seems most likely that there are multiple initiation sites for recombination once homolog recognition has been achieved.     

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.     

A large inverted duplication allows homologous recombination between chromosomes heterozygous for the proximal t complex inversion

We have examined the molecular organization of a region of mouse chromosome 17 that allows homologous recombination between wild-type and t haplotype chromosomes across a large inversion. We have used a combination of genetic mapping of restriction fragment length polymorphisms, molecular characterization of cloned regions isolated on overlapping cosmids, and subchromosomal restriction mapping using the pulsed field gel technique. Our analyses show that the wild-type form of chromosome 17 contains an inverted duplication of an element of at least 650 kb that is present in only one copy in the t haplotype form. Two chromosomes, th45 and tAE5, arose by homologous recombination across the element that is present in both chromosomal variants in the same orientation.     

Physical map of the chromosomal region of Corynebacterium diphtheriae containing corynephage attachment sites attB1 and attB2.

The chromosome of Corynebacterium diphtheriae C7 was recently shown to contain two equivalent attachment sites (attB1 and attB2) for lysogenization by corynephages (R. Rappuoli, J.L. Michel, and J.R. Murphy, J. Bacteriol. 153:1202-1210, 1983). Portions of bacterial chromosome containing each attB site, as well as a 3.5-kilobase (kb) EcoRI fragment containing both attB1 and attB2 sites, were cloned in the pUC8 plasmid vector. Restriction endonuclease mapping and Southern blot hybridization analysis of restriction endonuclease fragments showed that attB1 and attB2 are 2.25 kb apart on the chromosome. Furthermore, a 0.85-kb HincII-EcoRI restriction endonuclease fragment containing attB1, a 0.77-kb HincII-BamHI fragment containing attB2, and a 1.2-kb EcoRI-BamHI fragment containing attP share short homologous regions. No homology was detected between the sequences flanking the two attB sites. The isolation of a segregant which had lost the entire chromosomal segment contained between attB1 and attB2 suggests that this region is not essential for growth.     

Integration of linear, heterologous DNA molecules into the Bacillus subtilis chromosome: mechanism and use in induction of predictable rearrangements.

Linear DNA molecules composed of a central region nonhomologous with the Bacillus subtilis chromosome and two flanking regions homologous with the chromosome can integrate into the chromosome, provided that the homologous regions have the same relative orientation. The resulting chromosome can be maintained in a haploid or in a merodiploid cell together with a parental chromosome. This can most easily be explained by supposing that the integration occurs by crossing over at each homologous region and that a part of the chromosome between these regions is deleted and replaced by the central nonhomologous region of the integrating molecule. If no essential genes were replaced during that process a haploid cell would be obtained; if essential genes were replaced a merodiploid cell would be obtained. The use of appropriate linear molecules therefore should allow the induction of deletions, extending from a given chromosomal site in a predetermined direction, and defined duplications in the B. subtilis chromosome.     

Opal suppressor phosphoserine tRNA gene and pseudogene are located on human chromosomes 19 and 22, respectively

An opal suppressor phosphoserine tRNA gene and pseudogene have been isolated from a human DNA library and sequenced (O'Neill, V., Eden, F., Pratt, K., and Hatfield, D. (1985) J. Biol. Chem. 260, 2501-2508). Southern hybridization of human genomic DNA with an opal suppressor tRNA probe suggested that the gene and pseudogene are present in single copy. In this study, we have determined the chromosome location of the human gene and pseudogene by utilizing a 193-base pair fragment encoding the opal suppressor phosphoserine tRNA gene as probe to examine DNAs isolated from human-rodent somatic cell hybrids that have segregated human chromosomes. These studies show that the probe hybridized with two regions in the human genome; one is located on chromosome 19 and the second on chromosome 22. By comparing the restriction sites within these two regions to those previously determined for the human opal suppressor phosphoserine tRNA gene and pseudogene, we tentatively assigned the gene to chromosome 19 and the pseudogene to chromosome 22. These assignments were confirmed by utilizing a 350-base pair fragment which was isolated from the 5'-flanking region of the human gene as probe. This fragment hybridized only to chromosome 19, demonstrating unequivocally that the opal suppressor phosphoserine tRNA gene is located on chromosome 19. The flanking probe hybridized to a single homologous band in hamster and in mouse DNA to which the gene probe also hybridized, demonstrating that the 5'-flanking region of the opal suppressor tRNA gene is conserved in mammals. Restriction analysis of DNAs obtained from the white blood cells of 10 separate individuals demonstrates that the gene is polymorphic. This study provides two additional markers for the human genome and constitutes only the second set of two tRNA genes assigned to human chromosomes.     

Synaptonemal complex spreading in Allium cepa and A. fistulosum

The general features and fine structure of homologous chromosome alignment and pairing have been investigated in two species of Allium (A. fistulosum and A. cepa), which have similar karyotypes but very different patterns of chiasma distribution. Although there is no support for the occurrence of a general pre-meiotic alignment of homologous chromosomes, both species show some alignment of homologues as an immediate prelude to synaptonemal complex (SC) formation. In both species pairing usually commences at sub-terminal sites and is succeeded by numerous separate intercalary initiations of pairing in interstitial and distal regions and then in proximal regions. The last parts to pair, in both species, are pericentromeric and telomeric regions. There is, therefore, no evident relationship between the sequence of pairing and chiasma distribution in these species. Regularly alternating convergences and divergences of aligned axial cores (ACs), termed multiple association sites, are frequently observed. It is proposed that these represent potential pairing initiation sites and from observations on their spatial distribution it is argued that they may be evenly distributed through most of the genome. Small spherical or ellipsoid nodules are found at association sites and between closely aligned ACs which persist in the SC segments present during zygotene, but most of them disappear abruptly at the end of zygotene. These are termed zygotene nodules (ZN) and it is proposed that they are involved in matching corresponding sites on homologous chromosomes as well as possibly having a recombinational role. Their composition, structure, mode of action and relationship to pachytene recombination nodules are at present unknown.     

Differing requirements for RAD51 and DMC1 in meiotic pairing of centromeres and chromosome arms in Arabidopsis thaliana

During meiosis homologous chromosomes pair, recombine, and synapse, thus ensuring accurate chromosome segregation and the halving of ploidy necessary for gametogenesis. The processes permitting a chromosome to pair only with its homologue are not fully understood, but successful pairing of homologous chromosomes is tightly linked to recombination. In Arabidopsis thaliana, meiotic prophase of rad51, xrcc3, and rad51C mutants appears normal up to the zygotene/pachytene stage, after which the genome fragments, leading to sterility. To better understand the relationship between recombination and chromosome pairing, we have analysed meiotic chromosome pairing in these and in dmc1 mutant lines. Our data show a differing requirement for these proteins in pairing of centromeric regions and chromosome arms. No homologous pairing of mid-arm or distal regions was observed in rad51, xrcc3, and rad51C mutants. However, homologous centromeres do pair in these mutants and we show that this does depend upon recombination, principally on DMC1. This centromere pairing extends well beyond the heterochromatic centromere region and, surprisingly, does not require XRCC3 and RAD51C. In addition to clarifying and bringing the roles of centromeres in meiotic synapsis to the fore, this analysis thus separates the roles in meiotic synapsis of DMC1 and RAD51 and the meiotic RAD51 paralogs, XRCC3 and RAD51C, with respect to different chromosome domains.     

A chromosome‐level genome assembly of the parasitoid wasp Pteromalus puparum

Parasitoid wasps represent a large proportion of hymenopteran species. They have complex evolutionary histories and are important biocontrol agents. To advance parasitoid research, a combination of Illumina short‐read, PacBio long‐read and Hi‐C scaffolding technologies was used to develop a high‐quality chromosome‐level genome assembly for Pteromalus puparum, which is an important pupal endoparasitoid of caterpillar pests. The chromosome‐level assembly has aided in studies of venom and detoxification genes. The assembled genome size is 338 Mb with a contig N50 of 38.7 kb and a scaffold N50 of 1.16 Mb. Hi‐C analysis assembled scaffolds onto five chromosomes and raised the scaffold N50 to 65.8 Mb, with more than 96% of assembled bases located on chromosomes. Gene annotation was assisted by RNA sequencing for the two sexes and four different life stages. Analysis detected 98% of the BUSCO (Benchmarking Universal Single‐Copy Orthologs) gene set, supporting a high‐quality assembly and annotation. In total, 40.1% (135.6 Mb) of the assembly is composed of repetitive sequences, and 14,946 protein‐coding genes were identified. Although venom genes play important roles in parasitoid biology, their spatial distribution on chromosomes was poorly understood. Mapping has revealed venom gene tandem arrays for serine proteases, pancreatic lipase‐related proteins and kynurenine–oxoglutarate transaminases, which have amplified in the P. puparum lineage after divergence from its common ancestor with Nasonia vitripennis. In addition, there is a large expansion of P450 genes in P. puparum. These examples illustrate how chromosome‐level genome assembly can provide a valuable resource for molecular, evolutionary and biocontrol studies of parasitoid wasps.     

Dynamics of homologous chromosome pairing during meiotic prophase in fission yeast

Pairing of homologous chromosomes is important for homologous recombination and correct chromosome segregation during meiosis. It has been proposed that telomere clustering, nuclear oscillation, and recombination during meiotic prophase facilitate homologous chromosome pairing in fission yeast. Here we examined the contributions of these chromosomal events to homologous chromosome pairing, by directly observing the dynamics of chromosomal loci in living cells of fission yeast. Homologous loci exhibited a dynamic process of association and dissociation during the time course of meiotic prophase. Lack of nuclear oscillation reduced association frequency for both centromeric and arm regions of the chromosome. Lack of telomere clustering or recombination reduced association frequency at arm regions, but not significantly at centromeric regions. Our results indicate that homologous chromosomes are spatially aligned by oscillation of telomere-bundled chromosomes and physically linked by recombination at chromosome arm regions; this recombination is not required for association of homologous centromeres.     

Chloroplast DNA variation in pearl millet and related species

The evolution of specific regions of the chloroplast genome was studied in five grass species in the genus Pennisetum, including pearl millet, and one species from a related genus (Cenchrus). Three different regions of the chloroplast DNA were investigated. The first region included a 12-kilobase pair (kbp) EcoRI fragment containing the 23S, 16S and 5S ribosomal RNA genes, which is part of a larger duplicated region of reverse orientation. The second region was contained in a 21-kbp Sa/I fragment, which spans the short single-copy sequence separating the two reverse repeat structures and which overlaps the duplicated copies of the 12-kbp Eco RI fragment. The third region was a 6-kbp EcoRI fragment located in the large single-copy region of the chloroplast genome. Together these regions account for slightly less than 25% of the chloroplast genome. Each of these DNA fragments was cloned and used as hybridization probes to determine the distribution of homologous DNA fragments generated by various restriction endonuclease digests.—A survey of 12 geographically diverse collections of pearl millet showed no indication of chloroplast DNA sequence polymorphism, despite moderate levels of nuclear-encoded enzyme polymorphism. Interspecific and intergeneric differences were found for restriction endonuclease sites in both the small and the large single-copy regions of the chloroplast genome. The reverse repeat structure showed identical restriction site distributions in all materials surveyed. These results suggest that the reverse repeat region is differentially conserved during the evolution of the chloroplast genome.