Genomic structure and paralogous regions of the inversion breakpoint occurring between human chromosome 3p12.3 and orangutan chromosome 2

Intrachromosomal duplications play a significant role in human genome pathology and evolution. To better understand the molecular basis of evolutionary chromosome rearrangements, we performed molecular cytogenetic and sequence analyses of the breakpoint region that distinguishes human chromosome 3p12.3 and orangutan chromosome 2. FISH with region-specific BAC clones demonstrated that the breakpoint-flanking sequences are duplicated intrachromosomally on orangutan 2 and human 3q21 as well as at many pericentromeric and subtelomeric sites throughout the genomes. Breakage and rearrangement of the human 3p12.3-homologous region in the orangutan lineage were associated with a partial loss of duplicated sequences in the breakpoint region. Consistent with our FISH mapping results, computational analysis of the human chromosome 3 genomic sequence revealed three 3p12.3-paralogous sequence blocks on human chromosome 3q21 and smaller blocks on the short arm end 3p26-->p25. This is consistent with the view that sequences from an ancestral site at 3q21 were duplicated at 3p12.3 in a common ancestor of orangutan and humans. Our results show that evolutionary chromosome rearrangements are associated with microduplications and microdeletions, contributing to the DNA differences between closely related species.     

Segmental duplication associated with evolutionary instability of human chromosome 3p25.1

Fluorescence in situ hybridization (FISH) of human bacterial artificial chromosome (BAC) clones to orangutan metaphase spreads localized a breakpoint between human chromosome 3p25.1 and orangutan chromosome 2 to a <30-kb interval. The inversion occurred in a relatively gene-rich region with seven genes within 500 kb. The underlying breakpoint is closely juxtaposed to validated genes, however no functional gene has been disrupted by the evolutionary rearrangement. An approximately 21-kb DNA segment at the 3p25.1 breakpoint region has been duplicated intrachromosomally and interchromosomally to multiple regions in the orangutan and human genomes, providing additional evidence for the role of segmental duplications in hominoid chromosome evolution.     

Assignement of the fibronectin (FN1) gene to pongo pygmaeus (Organgutan) chromosome 11

Using Fibronectin (F18) human cDNA probe, the FN gene was assigned to orangutan chromosome 11 by in situ hybridization. Our data confirm the homology between orangutan chromosome 11 and the long arm of human chromosome 2.     

Comparative cytogenetics of human chromosome 3q21.3 reveals a hot spot for ectopic recombination in hominoid evolution

Fluorescence in situ hybridization mapping of fully integrated human BAC clones to primate chromosomes, combined with precise breakpoint localization by PCR analysis of flow-sorted chromosomes, was used to analyze the evolutionary rearrangements of the human 3q21.3-syntenic region in orangutan, siamang gibbon, and silvered-leaf monkey. Three independent evolutionary breakpoints were localized within a 230-kb segment contained in BACs RP11-93K22 and RP11-77P16. Approximately 200 kb of the human 3q21.3 sequence was not present on the homologous orangutan, siamang, and Old World monkey chromosomes, suggesting a genomic DNA insertion into the breakpoint region in the lineage leading to humans and African great apes. The breakpoints in the orangutan and siamang genomes were narrowed down to 12- and 20-kb DNA segments, respectively, which are enriched with endogenous retrovirus long terminal repeats and other repetitive elements. The inserted DNA segment represents part of an ancestral duplication. Paralogous sequence blocks were found at human 3q21, approximately 4 Mb proximal to the evolutionary breakpoint cluster region; at human 3p12.3, which contains an independent orangutan-specific breakpoint; and at the subtelomeric and pericentromeric regions of multiple human and orangutan chromosomes. The evolutionary breakpoint regions between human chromosome 3 and orangutan 2 as well their paralogous segments in the human genome coincide with breaks of chromosomal synteny in the mouse, rat, and/or chicken genomes. Collectively our data reveal reuse of the same short recombinogenic DNA segments in primate and vertebrate evolution, supporting a nonrandom breakage model of genome evolution.     

A human moderately repeated Y-specific DNA sequence is evolutionarily conserved in the Y chromosome of the great apes.

Evolutionary conservation of the human-derived moderately repeated Y-specific DNA sequence Y-190 (DYZ5) was investigated in the chimpanzee, orangutan, and gorilla. Southern blot analysis showed the presence of the sequence in the Y chromosome of all great apes. Pulsed-field gel electrophoresis and in situ hybridization revealed that the repeat is organized in one major block and confined to a small region of the Y chromosome of the three species. DYZ5 was assigned to the proximal short arm of the Y chromosome of the chimpanzee and orangutan and to the long arm of the Y chromosome of the gorilla. In light of its evolutionary conservation, DYZ5 may have an as yet undetermined structural function in the Y chromosome.     

Chromosome studies in the orangutan (Pongo pygmaeus): Practical applications for breeding and conservation

The unequivocal identification of Bornean, Sumatran, and first-generation hybrid orangutans can be carried out by chromosome analysis, a procedure that is more reliable than any other so far used to distinguish between orangutan subspecies. Chromosome differences between subspecies have been compared with protein and DNA studies, and these have shown that Bornean and Sumatran orangutans are more different from each other than we originally thought. Chromosome studies in the orangutan have shown variant chromosome types that are not subspecies-specific. One of these variant types is a product of a complex double inversion rearrangement and is a polymorphic trait in both subspecies. In view of our findings, specific guidelines have been recommended for evaluating the fertility of hybrid specimens and maintaining purebred orangutan stocks.     

Plasticity of human chromosome 3 during primate evolution

Comparative mapping of more than 100 region-specific clones from human chromosome 3 in Bornean and Sumatran orangutans, siamang gibbon, and Old and New World monkeys allowed us to reconstruct ancestral simian and hominoid chromosomes. A single paracentric inversion derives chromosome 1 of the Old World monkey Presbytis cristata from the simian ancestor. In the New World monkey Callithrix geoffroyi and siamang, the ancestor diverged on multiple chromosomes, through utilizing different breakpoints. One shared and two independent inversions derive Bornean orangutan 2 and human 3, implying that neither Bornean orangutans nor humans have conserved the ancestral chromosome form. The inversions, fissions, and translocations in the five species analyzed involve at least 14 different evolutionary breakpoints along the entire length of human 3; however, particular regions appear to be more susceptible to chromosome reshuffling. The ancestral pericentromeric region has promoted both large-scale and micro-rearrangements. Small segments homologous to human 3q11.2 and 3q21.2 were repositioned intrachromosomally independent of the surrounding markers in the orangutan lineage. Breakage and rearrangement of the human 3p12.3 region were associated with extensive intragenomic duplications at multiple orangutan and gibbon subtelomeric sites. We propose that new chromosomes and genomes arise through large-scale rearrangements of evolutionarily conserved genomic building blocks and additional duplication, amplification, and/or repositioning of inherently unstable smaller DNA segments contained within them.     

"Bar-coding" primate chromosomes: molecular cytogenetic screening for the ancestral hominoid karyotype

Two recently introduced multicolor FISH approaches, cross-species color banding (also termed Rx-FISH) and multiplex FISH using painting probes derived from somatic cell hybrids retaining fragments of human chromosomes, were applied in a comparative molecular cytogenetic study of higher primates. We analyzed these "chromosome bar code" patterns to obtain an overview of chromosomal rearrangements that occurred during higher primate evolution. The objective was to reconstruct the ancestral genome organization of hominoids using the macaque as outgroup species. Approximately 160 individual and discernible molecular cytogenetic markers were assigned in these species. Resulting comparative maps allowed us to identify numerous intra-chromosomal rearrangements, to discriminate them from previous contradicting chromosome banding interpretations and to propose an ancestral karyotype for hominoids. From 25 different chromosome forms in an ancestral karyotype for all hominoids of 2N=48 we propose 21. Probes for chromosomes 2p, 4, 9 and Y were not informative in the present experiments. The orangutan karyotype was very similar to the proposed ancestral organization and conserved 19 of the 21 ancestral forms; thus most chromosomes were already present in early hominoid evolution, while African apes and human show various derived changes.     

Fluorescent (F) bodies in the spermatozoa of man and the great apes

Mature spermatozoa of the chimpanzee (Pan troglodytes), the gorilla (Gorilla gorilla), and the orangutan (Pongo pygmaeus) were stained with quinacrine dihydrochloride. Fluorescent (F) bodies were visualized in the spermatozoa of the chimpanzee and gorilla but were absent in the orangutan, in which there is no brilliant fluorescence in any chromosome. The F bodies appeared to be randomly located in the sperm heads of these two species, as they usually are in human spermatozoa. However, the proportion of sperm showing one or more F bodies in the chimpanzee and gorilla was not comparable to what is usually found in man. The F bodies in the chimpanzee presumably represent brilliant regions in the autosomes, since the Y chromosome has no brilliant fluorescence in this species. This is contrary to man, in which the F body is an useful indicator of the Y chromosome. In the gorilla, the F bodies probably correspond to both the Y chromosome and to some brilliant regions in the autosomes.     

Detection of nucleolus organizer regions in chromosomes of human,chimpanzee, gorilla,orangutan and gibbon

Nucleolus organizer regions were detected by the Ag-AS silver method in fixed metaphase chromosomes from human and primates. In the human, silver was deposited in the secondary constriction of a maximum of five pairs of acrocentric chromosomes: 13, 14, 15, 21 and 22. The chimpanzee also had five pairs of acrocentric chromosomes stained, corresponding to human numbers 13, 14, 18, 21 and 22. A gibbon had a single pair of chromosomes with a secondary constriction, which corresponded to the nucleolus organizer region. In each case the Ag-AS method detected the sites which have been shown by in situ hybridization to contain the ribosomal RNA genes. An orangutan had eight pairs of acrocentric chromosomes stained with Ag-AS, probably corresponding to human numbers 13, 14, 15, 18, 21 and 22, plus two others. Two gorillas had silver stain over two pairs of small acrocentric chromosomes and at the telomere of one chromosome 1. The larger gorilla acrocentric chromosomes had no silver stain although they all had secondary constrictions and entered into satellite associations.     

Regional chromosomal assignment of the human mineralocorticoid receptor gene to 4q31.1

Summary The gene for human mineralocorticoid receptor (hMR), previously mapped to chromosome 4, has been further localized to 4q31.1 by in situ hybridization using a biotinylated 3.75kb human cDNA clone encoding the primary amino acid sequence of hMR as a probe. Preliminary comparative mapping studies in orangutan (Pongo pygmaeus) suggest localization of the probe to the long arm of chromosome 3.     

Panels of somatic cell hybrids specific for chimpanzee, gorilla, orangutan, and baboon

The generation of panels of somatic cell hybrids specific for chimpanzee, gorilla, orangutan, and olive baboon is reported. The chromosome content of each hybrid clone was characterized using reverse painting on human normal metaphases and by the use of appropriate sequence tag sites (STSs), one for each chromosome arm. These resources can be advantageously exploited in the characterization of chromosome architecture of different primate species, with special reference to the discrimination of inter- and intra-chromosomal arrangement of segmental duplications.     

Cytochrome c oxidase assembly in primates is sensitive to small evolutionary variations in amino acid sequence

Respiring mitochondria require many interactions between nuclear and mitochondrial genomes. Although mitochondrial DNA (mtDNA) from the gorilla and the chimpanzee are able to restore oxidative phosphorylation in a human cell, mtDNAs from more distant primate species are functionally incompatible with human nuclear genes. Using microcell-mediated chromosome and mitochondria transfer, we introduced and maintained a functional orangutan mtDNA in a human nuclear background. However, partial oxidative phosphorylation function was restored only in the presence of most orangutan chromosomes, suggesting that human oxidative phosphorylation-related nuclear-coded genes are not able to replace many orangutan ones. The respiratory capacity of these hybrids was decreased by 65%-80%, and cytochrome c oxidase (COX) activity was decreased by 85%-95%. The function of other respiratory complexes was not significantly altered. The translation of mtDNA-coded COX subunits was normal, but their steady-state levels were approximately 10% of normal ones. Nuclear-coded COX subunits were loosely associated with mitochondrial membranes, a characteristic of COX assembly-defective mutants. Our results suggest that many human nuclear-coded genes not only cannot replace the orangutan counterparts, but also exert a specific interference at the level of COX assembly. This cellular model underscores the precision of COX assembly in mammals and sheds light on the nature of nuclear-mtDNA coevolutionary constraints.     

A Y-chromosomal DNA fragment is conserved in human and chimpanzee.

A human male-specific Y-chromosomal DNA fragment (lambda YH2D6) has been isolated. By deletion-mapping analysis, 2D6 has been localized to the euchromatic portion of the long arm (Yq11) of the human Y chromosome. Among great apes, this fragment was found to be conserved in male chimpanzee but was lacking in male gorilla and male orangutan. No homologous fragments were detected in females of orangutan, gorilla, chimpanzee, or human. Nucleotide sequence analysis indicated the presence of partial-Alu-elements and of sequences similar to the GATA repeats of the snake Bkm sequence.     

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.     

Evolution of beta satellite DNA sequences: evidence for duplication-mediated repeat amplification and spreading

In this article, we report studies on the evolutionary history of beta satellite repeats (BSR) in primates. In the orangutan genome, the bulk of BSR sequences was found organized as very short stretches of approximately 100 to 170 bp, embedded in a 60-kb to 80-kb duplicated DNA segment. The estimated copy number of the duplicon that carries BSR sequences ranges from 70 to 100 per orangutan haploid genome. In both macaque and gibbon, the duplicon mapped to a single chromosomal region at the boundary of the rDNA on the marker chromosome (chromosome 13 and 12, respectively). However, only in the gibbon, the duplicon comprised 100 bp of beta satellite. Thus, the ancestral copy of the duplicon appeared in Old World monkeys ( approximately 25 to approximately 35 MYA), whereas the prototype of beta satellite repeats took place in a gibbon ancestor, after apes/Old World monkeys divergence ( approximately 25 MYA). Subsequently, a burst in spreading of the duplicon that carries the beta satellite was observed in the orangutan, after lesser apes divergence from the great apes-humans lineage ( approximately 18 MYA). The analysis of the orangutan genome also indicated the existence of two variants of the duplication that differ for the length (100 or 170 bp) of beta satellite repeats. The latter organization was probably generated by nonhomologous recombination between two 100-bp repeated regions, and it likely led to the duplication of the single Sau3A site present in the 100-bp variant, which generated the prototype of Sau3A 68-bp beta satellite tandem organization. The two variants of the duplication, although with a different ratios, characterize the hominoid genomes from the orangutan to humans, preferentially involving acrocentric chromosomes. At variance to alpha satellite, which appeared before the divergence of New World and Old World monkeys, the beta satellite evolutionary history began in apes ancestor, where we have first documented a low-copy, nonduplicated BSR sequence. The first step of BSR amplification and spreading occurred, most likely, because the BSR was part of a large duplicon, which underwent a burst dispersal in great apes' ancestor after the lesser apes' branching. Then, after orangutan divergence, BSR acquired the clustered structural organization typical of satellite DNA.     

A human-derived probe,p82H,hybridizes to the centromeres of gorilla,chimpanzee, and orangutan

A human-derived centromeric sequence, p82H, hybridizes to DNA from gorilla, chimpanzee, pygmy chimpanzee, and orangutan. On DNA blots, multimeric ladders based on 170 or 340 bp repeat units are seen. In metaphase chromosome preparations from these species, p82H hybridizes to the centromeric region of every chromosome. p82H forms less stable hybrids with DNA from the lion-tailed macaque and does not hybridize to DNA or chromosomes of the owl monkey or the mouse.     

Regional localization of human M-BCR gene to chromosome 23 band q11 in the great apes

We hybridized a human M-BCR DNA probe to the chromosomes of chimpanzee (Pan troglodytes), gorilla (Gorilla gorilld) and orangutan (Pongo pygmaeus) by FISH-technique. The human M-BCR gene was localized to chromosome 23 band q11 (23q11), which is equivalent to the human chromosome 22 band q11 in all three species. The conservation of M-BCR gene in higher primates at the corresponding human chromosome locus provides phylogenetic clues concerning the evolution of genes.     

The mitochondrial DNA molecule of sumatran orangutan and a molecular proposal for two (Bornean and Sumatran) species of orangutan

The complete mitochondrial DNA (mtDNA) molecule of Sumatran orangutan, plus the complete mitochondrial control region of another Sumatran specimen and the control regions and five protein-coding genes of two specimens of Bornean orangutan were sequenced and compared with a previously reported complete mtDNA of Bornean orangutan. The two orangutans are presently separated at the subspecies level. Comparison with five different species pairs—namely, harbor seal/grey seal, horse/donkey, fin whale/blue whale, common chimpanzee/pygmy chimpanzee, and Homo/common chimpanzee—showed that the molecular difference between Sumatran and Bornean orangutan is much greater than that between the seals, and greater than that between the two chimpanzees, but similar to that between the horse and the donkey and the fin and blue whales. Considering their limited morphological distinction the comparison revealed unexpectedly great molecular difference between the two orangutans. The nucleotide difference between the orangutans is about 75% of that between Homo and the common chimpanzee, whereas the amino acid difference exceeds that between Homo and the common chimpanzee. On the basis of their molecular distinction we propose that the two orangutans should be recognized as different species, Pongo pygmaeus, Bornean orangutan, and P. abelii, Sumatran orangutan. Received: 15 May 1996 / Accepted: 21 June 1996