A Siglec-like sialic-acid-binding motif revealed in an adenovirus capsid protein

Sialic-acid-binding immunoglobulin-like lectins (Siglecs) are a family of transmembrane receptors that are well documented to play roles in regulation of innate and adaptive immune responses. To see whether the features that define the molecular recognition of sialic acid were found in other sialic-acid-binding proteins, we analyzed 127 structures with bound sialic acids found in the Protein Data Bank database. Of these, the canine adenovirus 2-fiber knob protein showed close local structural relationship to Siglecs despite low sequence similarity. The fiber knob harbors a noncanonical sialic-acid recognition site, which was then explored for detailed specificity using a custom glycan microarray comprising 58 diverse sialosides. It was found that the adenoviral protein preferentially recognizes the epitope Neu5Acα2-3[6S]Galβ1-4GlcNAc, a structure previously identified as the preferred ligand for Siglec-8 in humans and Siglec-F in mice. Comparison of the Siglec and fiber knob sialic-acid-binding sites reveal conserved structural elements that are not clearly identifiable from the primary amino acid sequence, suggesting a Siglec-like sialic-acid-binding motif that comprises the consensus features of these proteins in complex with sialic acid.     

Cloning and characterization of a novel mouse Siglec, mSiglec-F: differential evolution of the mouse and human (CD33) Siglec-3-related gene clusters

A novel mouse Siglec (mSiglec-F) belonging to the subfamily of Siglec-3-related Siglecs has been cloned and characterized. Unlike most human Siglec-3 (hSiglec-3)-related Siglecs with promiscuous linkage specificity, mSiglec-F shows a strong preference for alpha2-3-linked sialic acids. It is predominantly expressed in immature cells of the myelomonocytic lineage and in a subset of CD11b (Mac-1)-positive cells in some tissues. As with previously cloned Siglec-3-related mSiglecs, the lack of strong sequence similarity to a singular hSiglec made identification of the human ortholog difficult. We therefore conducted a comprehensive comparison of Siglecs between the human and mouse genomes. The mouse genome contains eight Siglec genes, whereas the human genome contains 11 Siglec genes and a Siglec-like gene. Although a one-to-one orthologous correspondence between human and mouse Siglecs 1, 2, and 4 is confirmed, the Siglec-3-related Siglecs showed marked differences between human and mouse. We found only four Siglec genes and two pseudogenes in the mouse chromosome 7 region syntenic to the Siglec-3-related gene cluster on human chromosome 19, which, in contrast, contains seven Siglec genes, a Siglec-like gene, and thirteen pseudogenes. Although analysis of gene maps and exon structures allows tentative assignments of mouse-human Siglec ortholog pairs, the possibility of unequal genetic recombination makes the assignments inconclusive. We therefore support a temporary lettered nomenclature for additional mouse Siglecs. Current information suggests that mSiglec-F is likely a hSiglec-5 ortholog. The previously reported mSiglec-3/CD33 and mSiglec-E/MIS are likely orthologs of hSiglec-3 and hSiglec-9, respectively. The other Siglec-3-like gene in the cluster (mSiglec-G) is probably a hSiglec-10 ortholog. Another mouse gene (mSiglec-H), without an apparent human ortholog, lies outside of the cluster. Thus, although some duplications of Siglec-3-related genes predated separation of the primate and rodent lineages (about 80-100 million years ago), this gene cluster underwent extensive duplications in the primate lineage thereafter.     

Cloning and characterization of human Siglec-11. A recently evolved signaling molecule that can interact with SHP-1 and SHP-2 and is expressed by tissue macrophages, including brain microglia

Siglecs are sialic acid-recognizing animal lectins of the immunoglobulin superfamily. We have cloned and characterized a novel human molecule, Siglec-11, that belongs to the subgroup of CD33/Siglec-3-related Siglecs. As with others in this subgroup, the cytosolic domain of Siglec-11 is phosphorylated at tyrosine residue(s) upon pervanadate treatment of cells and then recruits the protein-tyrosine phosphatases SHP-1 and SHP-2. However, Siglec-11 has several novel features relative to the other CD33/Siglec-3-related Siglecs. First, it binds specifically to alpha2-8-linked sialic acids. Second, unlike other CD33/Siglec-3-related Siglecs, Siglec-11 was not found on peripheral blood leukocytes. Instead, we observed its expression on macrophages in various tissues, such as liver Kupffer cells. Third, it was also expressed on brain microglia, thus becoming the second Siglec to be found in the nervous system. Fourth, whereas the Siglec-11 gene is on human chromosome 19, it lies outside the previously described CD33/Siglec-3-related Siglec cluster on this chromosome. Fifth, analyses of genome data bases indicate that Siglec-11 has no mouse ortholog and that it is likely to be the last canonical human Siglec to be reported. Finally, although Siglec-11 shows marked sequence similarity to human Siglec-10 in its extracellular domain, the cytosolic tail appears only distantly related. Analysis of genomic regions surrounding the Siglec-11 gene suggests that it is actually a chimeric molecule that arose from relatively recent gene duplication and recombination events, involving the extracellular domain of a closely related ancestral Siglec gene (which subsequently became a pseudogene) and a transmembrane and cytosolic tail derived from another ancestral Siglec.     

Siglec-7: a sialic acid-binding lectin of the immunoglobulin superfamily

The Siglecs are a recently discovered family of sialic acid-binding lectins of the immunoglobulin (Ig) superfamily. We report a molecule showing homology to the six first reported Siglecs, with the closest relationship to Siglec-3(CD33), Siglec-5, and Siglec-6(OBBP-1). The extracellular portion has two Ig-like domains, with the amino-terminal V-set Ig domain including amino acid residues known to be involved in sialic acid recognition by other Siglecs. The cytoplasmic domain has putative sites of tyrosine phosphorylation shared with some Siglecs, including an Immuno-receptor Tyrosine-based Inhibitory Motif (ITIM). Expression of the full-length cDNA induces sialic acid-dependent binding to human erythrocytes. A recombinant chimeric form containing the extracellular Ig domains selectively recognizes the sequence Neu5Acalpha2-6Galbeta1-4Glc, and binding requires the side chain of sialic acid. Mutation of an arginine residue predicted to be critical for sialic acid binding abolishes both interactions. Taken together, our findings justify designation of the molecule as Siglec-7. Analysis of bacterial artificial chromosome (BAC) clones spanning the known human genomic location of Siglec-3 indicates that the Siglec-7 gene is also located on chromosome 19q13.3-13.4. Human tissues show strong expression of Siglec-7 mRNA in spleen, peripheral blood leukocytes, and liver. The combination of an extracellular sialic acid binding site and an intracellular ITIM motif suggests that this molecule is involved in trans-membrane regulatory signaling reactions.     

Molecular characterization, tissue expression, and mapping of a novel Siglec-like gene (SLG2) with three splice variants.

The sialic acid binding immunglobulin-like lectin (Siglec) family is a recently described member of the immunoglobulin superfamily. Within the Siglec family, there exists a subgroup, which bears a high degree of homology with the molecule CD33 (Siglec-3), and has thus been designated the CD33-like subgroup of Siglecs. Members of this subgroup have been localized to chromosome 19q13.4. Through the positional candidate approach, we identified a novel potential member of this subgroup of Siglecs. We have characterized the complete genomic structure of this gene, determined its chromosomal localization, its homology to other members of the Siglec family, and its tissue expression profile. This new Siglec-like gene is comprised of 11 exons, with 10 intervening introns, and is localized 278 kb telomeric to Siglec-9 and 35 kb centromeric to Siglec-8 and on chromosome 19q13.4. The coding region consists of 2094 base pairs, and encodes for a putative 76.6 kDa protein. All Siglec-conserved structural features, including V-set domain, three C-set domains, transmembrane domain, ITIM and SLAM motifs, were found in this Siglec-like gene. Also, it has the conserved amino acids essential for sialic acid binding. The Siglec-like gene has 40-66% homology with members of the CD33-like subgroup, including Siglecs 5-9. Through RT-PCR we have examined the expression profile of this new gene in a panel of human tissues and found it to be primarily expressed in the bone marrow, spleen, brain, small intestine, colon, and spinal cord. We were also able to identify three different splice variants of the new gene. This gene may represent the latest novel member of the CD33-like subgroup of Siglecs, and, given its high degree of homology, it may also serve a regulatory role in the proliferation and survival of a particular hematopoietic stem cell lineage, as has been found for CD33 and Siglec-7.     

A structural difference between the cell surfaces of humans and the great apes

The sialic acids are major components of the cell surfaces of animals of the deuterostome lineage. Earlier studies suggested that humans may not express N-glycolyl-neuraminic acid (Neu5Gc), a hydroxylated form of the common sialic acid N-acetyl-neuraminic acid (Neu5Ac). We find that while Neu5Gc is essentially undetectable on human plasma proteins and erythrocytes, it is a major component in all the four extant great apes (chimpanzee, bonobo, gorilla and orangutan) as well as in many other mammals. This marked difference is also seen amongst cultured lymphoblastoid cells from humans and great apes, as well as in a variety of other tissues compared between humans and chimpanzees, including the cerebral cortex and the cerebrospinal fluid. Biosynthetically, Neu5Gc arises from the action of a hydroxylase that converts the nucleotide donor CMP-Neu5Ac to CMP-Neu5Gc. This enzymatic activity is present in chimpanzee cells, but not in human cells. However, traces of Neu5Gc occur in some human tissues, and others have reported expression of Neu5Gc in human cancers and fetal tissues. Thus, the enzymatic capacity to express Neu5Gc appears to have been suppressed sometime after the great ape-hominid divergence. As terminal structures on cell surfaces, sialic acids are involved in intercellular cross-talk involving specific vertebrate lectins, as well as in microbe-host recognition involving a wide variety of pathogens. The level of sialic acid hydroxylation (level of Neu5Ac versus Neu5Gc) is known to positively or negatively affect several of these endogenous and exogenous interactions. Thus, there are potential functional consequences of this widespread structural change in humans affecting the surfaces of cells throughout the body. Am J Phys Anthropol 107:187-198, 1998. © 1998 Wiley-Liss, Inc.     

Cloning, characterization, and phylogenetic analysis of siglec-9, a new member of the CD33-related group of siglecs. Evidence for co-evolution with sialic acid synthesis pathways

The Siglecs are a subfamily of I-type lectins (immunoglobulin superfamily proteins that bind sugars) that specifically recognize sialic acids. We report the cloning and characterization of human Siglec-9. The cDNA encodes a type 1 transmembrane protein with three extracellular immunoglobulin-like domains and a cytosolic tail containing two tyrosines, one within a typical immunoreceptor tyrosine-based inhibitory motif (ITIM). The N-terminal V-set Ig domain has most amino acid residues typical of Siglecs. Siglec-9 is expressed on granulocytes and monocytes. Expression of the full-length cDNA in COS cells induces sialic-acid dependent erythrocyte binding. A recombinant soluble form of the extracellular domain binds to alpha2-3 and alpha2-6-linked sialic acids. Typical of Siglecs, the carboxyl group and side chain of sialic acid are essential for recognition, and mutation of a critical arginine residue in domain 1 abrogates binding. The underlying glycan structure also affects binding, with Galbeta1-4Glc[NAc] being preferred. Siglec-9 shows closest homology to Siglec-7 and both belong to a Siglec-3/CD33-related subset of Siglecs (with Siglecs-5, -6, and -8). The Siglec-9 gene is on chromosome 19q13.3-13.4, in a cluster with all Siglec-3/CD33-related Siglec genes, suggesting their origin by gene duplications. A homology search of the Drosophila melanogaster and Caenorhabditis elegans genomes suggests that Siglec expression may be limited to animals of deuterostome lineage, coincident with the appearance of the genes of the sialic acid biosynthetic pathway.     

Loss of N-glycolylneuraminic acid in human evolution. Implications for sialic acid recognition by siglecs

The common sialic acids of mammalian cells are N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Humans are an exception, because of a mutation in CMP-sialic acid hydroxylase, which occurred after our common ancestor with great apes. We asked if the resulting loss of Neu5Gc and increase in Neu5Ac in humans alters the biology of the siglecs, which are Ig superfamily members that recognize sialic acids. Human siglec-1 (sialoadhesin) strongly prefers Neu5Ac over Neu5Gc. Thus, humans have a higher density of siglec-1 ligands than great apes. Siglec-1-positive macrophages in humans are found primarily in the perifollicular zone, whereas in chimpanzees they also occur in the marginal zone and surrounding the periarteriolar lymphocyte sheaths. Although only a subset of chimpanzee macrophages express siglec-1, most human macrophages are positive. A known evolutionary difference is the strong preference of mouse siglec-2 (CD22) for Neu5Gc, contrasting with human siglec-2, which binds Neu5Ac equally well. To ask when the preference for Neu5Gc was adjusted in the human lineage, we cloned the first three extracellular domains of siglec-2 from all of the great apes and examined their preference. In fact, siglec-2 had evolved a higher degree of recognition flexibility before Neu5Gc was lost in humans. Human siglec-3 (CD33) and siglec-6 (obesity-binding protein 1) also recognize both Neu5Ac and Neu5Gc, and siglec-5 may have some preference for Neu5Gc. Others showed that siglec-4a (myelin-associated glycoprotein) prefers Neu5Ac over Neu5Gc. Thus, the human loss of Neu5Gc may alter biological processes involving siglec-1, and possibly, siglec-4a or -5.     

A uniquely human consequence of domain-specific functional adaptation in a sialic acid-binding receptor

Most mammalian cell surfaces display two major sialic acids (Sias), N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Humans lack Neu5Gc due to a mutation in CMP-Neu5Ac hydroxylase, which occurred after evolutionary divergence from great apes. We describe an apparent consequence of human Neu5Gc loss: domain-specific functional adaptation of Siglec-9, a member of the family of sialic acid-binding receptors of innate immune cells designated the CD33-related Siglecs (CD33rSiglecs). Binding studies on recombinant human Siglec-9 show recognition of both Neu5Ac and Neu5Gc. In striking contrast, chimpanzee and gorilla Siglec-9 strongly prefer binding Neu5Gc. Simultaneous probing of multiple endogenous CD33rSiglecs on circulating blood cells of human, chimp, or gorilla suggests that the binding differences observed for Siglec-9 are representative of multiple CD33rSiglecs. We conclude that Neu5Ac-binding ability of at least some human CD33rSiglecs is a derived state selected for following loss of Neu5Gc in the hominid lineage. These data also indicate that endogenous Sias (rather than surface Sias of bacterial pathogens) are the functional ligands of CD33rSiglecs and suggest that the endogenous Sia landscape is the major factor directing evolution of CD33rSiglec binding specificity. Exon-1-encoded Sia-recognizing domains of human and ape Siglec-9 share only approximately 93-95% amino acid identity. In contrast, the immediately adjacent intron and exon 2 have the approximately 98-100% identity typically observed among these species. Together, our findings suggest ongoing adaptive evolution specific to the Sia-binding domain, possibly of an episodic nature. Such domain-specific divergences should also be considered in upcoming comparisons of human and chimpanzee genomes.     

Siglec and anti-Siglec therapies

Siglecs (sialic acid–binding immunoglobulin-like lectins) are a family of receptors that bind sialic acids in specific linkages on glycoproteins and glycolipids. Siglecs play roles in immune signalling and exhibit cell-type specific expression and endocytic properties. Recent studies suggest that Siglecs are likely to function as immune checkpoints that regulate responses in cancers and inflammatory diseases. In this review, we discuss strategies to target the Siglec–sialic acid axis in human diseases, particularly cancer, and the possibility of exploiting them for therapeutic intervention.     

Inversion, duplication, and changes in gene context are associated with human chromosome 18 evolution

Human chromosome 18 differs from its homologues in the great apes by a pericentric inversion. We have identified a chimpanzee bacterial artificial chromosome that spans a region where a break is likely to have occurred in a human progenitor and have characterized the corresponding regions in both chimpanzees and humans. Interspecies sequence comparisons indicate that the ancestral break occurred between the genes ROCK1 and USP14. In humans, the inversion places ROCK1 near centromeric heterochromatin and USP14 adjacent to highly repetitive subtelomeric repeats. In addition, we provide evidence for a human segmental duplication that may have provided a mechanism for the inversion.     

O-acetylation and de-O-acetylation of sialic acids. Sialic acid esterases of diverse evolutionary origins have serine active sites and essential arginine residues

We and others have recently described 9-O-acetyl-sialic acid esterase (9-O-Ac-SA esterase) activities that appear to be specific for removal of O-acetyl esters from the 9-position of naturally occurring sialic acids. We have now examined a variety of species for such enzymes and found them in vertebrates and higher invertebrates, but not in plants or in lower invertebrates. This evolutionary distribution correlates well with that of the sialic acids themselves. All of the 9-O-Ac-SA esterase activities tested were inhibited by diisopropyl fluorophosphate (DFP) in a dose-dependent fashion. This indicates that each of these enzymes has a serine active site similar to the well known serine esterases and serine proteases. Methyl esterification of the carboxyl group of 9-O-acetyl-N-acetylneuraminic acid significantly reduced the activity of all of the 9-O-Ac-SA esterases against the O-acetyl group. This indicates that each of these enzymes may recognize the negatively charged carboxyl group of the sialic acid. Enzymes that recognize anionic substrates frequently have an essential arginine residue (Riordan, J. F., McElvany, K. D., and Borders, C. L., Jr. (1977) Science 195, 884-886). We therefore studied the effects of the arginine-specific modifying reagents 2,3-butanedione and phenylglyoxal on 9-O-Ac-SA esterase activities from influenza C virus, human erythrocytes, rat liver, starfish gonads, and sea bass brain. All of these enzymes were inhibited in a dose-dependent fashion by both reagents, under conditions previously known to avoid nonspecific modification. In contrast, the typical serine proteases trypsin and kallikrein and the serine esterase acetylcholinesterase were not significantly affected, even by the highest concentrations of these reagents used. These data indicate that five 9-O-Ac-SA esterase activities from evolutionarily distinct origins all have serine active sites and essential arginine residues. We postulate that the arginine residue is involved in substrate recognition via the negatively charged carboxyl group of the sialic acids. Thus, these 9-O-Ac-SA esterase activities may be members of a previously undescribed class of serine esterase.     

Multiple changes in sialic acid biology during human evolution

Humans are genetically very similar to “great apes”, (chimpanzees, bonobos, gorillas and orangutans), our closest evolutionary relatives. We have discovered multiple genetic and biochemical differences between humans and these other hominids, in relation to sialic acids and in Siglecs (Sia-recognizing Ig superfamily lectins). An inactivating mutation in the CMAH gene eliminated human expression of N-glycolylneuraminic acid (Neu5Gc) a major sialic acid in “great apes”. Additional human-specific changes have been found, affecting at least 10 of the <60 genes known to be involved in the biology of sialic acids. There are potential implications for unique features of humans, as well as for human susceptibility or resistance to disease. Additionally, metabolic incorporation of Neu5Gc from animal-derived materials occurs into biotherapeutic molecules and cellular preparations - and into human tissues from dietary sources, particularly red meat and milk products. As humans also have varying and sometime high levels of circulating anti-Neu5Gc antibodies, there are implications for biotechnology products, and for some human diseases associated with chronic inflammation.     

Cloning and characterization of Siglec-10, a novel sialic acid binding member of the Ig superfamily, from human dendritic cells

The Siglecs (sialic acid-binding Ig-like lectins) are a subfamily of I-type lectins, which specifically recognize sialic acids. Nine members of the family have been identified thus far. We have obtained a novel cDNA clone from a human dendritic cell cDNA library encoding a protein with sequence and structural features of the Siglec family, hence designated as Siglec-10. The full-length Siglec-10 cDNA encodes a type 1 transmembrane protein containing four extracellular immunoglobulin-like domains, a transmembrane region, and a cytoplasmic tail with two classical immunoreceptor tyrosine-based inhibitory motifs. The N-terminal V-set Ig domain has most of the amino acid residues typical of the Siglecs. Siglec-10 shows the closest homology to Siglec-5 and Siglec-3/CD33. Various cells and cell lines including monocytes and dendritic cells express Siglec-10. High levels of mRNA expression were seen in peripheral blood leukocytes, spleen, and liver. When expressed on COS-7 cells, Siglec-10 was able to bind human red blood cells and soluble sialoglycoconjugates in a sialic acid-dependent manner. The identification of Siglec-10 as a new Siglec family member and its expression profile, together with its sialic acid-dependent binding capacity, suggest that it may be involved in cell-cell recognition by interacting with sialylated ligands expressed on specific cell populations.     

Characterization of a Novel Class of Interspersed LTR Elements in Primate Genomes: Structure, Genomic Distribution, and Evolution

Retrovirus-like sequences and their solitary (solo) long terminal repeats (LTRs) are common repetitive elements in eukaryotic genomes. We reported previously that the tandemly arrayed genes encoding U2 snRNA (the RNU2 locus) in humans and apes contain a solo LTR (U2-LTR) which was presumably generated by homologous recombination between the two LTRs of an ancestral provirus that is retained in the orthologous baboon RNU2 locus. We have now sequenced the orthologous U2-LTRs in human, chimpanzee, gorilla, orangutan, and baboon and examined numerous homologs of the U2-LTR that are dispersed throughout the human genome. Although these U2-LTR homologs have been collectively referred to as LTR13 in the literature, they do not display sequence similarity to any known retroviral LTRs; however, the structure of LTR13 closely resembles that of other retroviral LTRs with a putative promoter, polyadenylation signal, and a tandemly repeated 53-bp enhancer-like element. Genomic blotting indicates that LTR13 is primate-specific; based on sequence analysis, we estimate there are about 2,500 LTR13 elements in the human genome. Comparison of the primate U2-LTR sequences suggests that the homologous recombination event that gave rise to the solo U2-LTR occurred soon after insertion of the ancestral provirus into the ancestral U2 tandem array. Phylogenetic analysis of the LTR13 family confirms that it is diverse, but the orthologous U2-LTRs form a coherent group in which chimpanzee is closest to the humans; orangutan is a clear outgroup of human, chimpanzee, and gorilla; and baboon is a distant relative of human, chimpanzee, gorilla, and orangutan. We compare the LTR13 family with other known LTRs and consider whether these LTRs might play a role in concerted evolution of the primate RNU2 locus. Received: 29 September 1997 / Accepted: 16 January 1998     

SIGLEC12, a human-specific segregating (pseudo)gene, encodes a signaling molecule expressed in prostate carcinomas

The primate SIGLEC12 gene encodes one of the CD33-related Siglec family of signaling molecules in immune cells. We had previously reported that this gene harbors a human-specific missense mutation of the codon for an Arg residue required for sialic acid recognition. Here we show that this R122C mutation of the Siglec-XII protein is fixed in the human population, i.e. it occurred prior to the origin of modern humans. Additional mutations have since completely inactivated the SIGLEC12 gene in some but not all humans. The most common inactivating mutation with a global allele frequency of 58% is a single nucleotide frameshift that markedly shortens the open reading frame. Unlike other CD33-related Siglecs that are primarily found on immune cells, we found that Siglec-XII protein is expressed not only on some macrophages but also on various epithelial cell surfaces in humans and chimpanzees. We also found expression on certain human prostate epithelial carcinomas and carcinoma cell lines. This expression correlates with the presence of the nonframeshifted, intact SIGLEC12 allele. Although SIGLEC12 allele status did not predict prostate carcinoma incidence, restoration of expression in a prostate carcinoma cell line homozygous for the frameshift mutation induced altered regulation of several genes associated with carcinoma progression. These stably transfected Siglec-XII-expressing prostate cancer cells also showed enhanced growth in nude mice. Finally, monoclonal antibodies against the protein were internalized by Siglec-XII-expressing prostate carcinoma cells, allowing targeting of a toxin to such cells. Polymorphic expression of Siglec-XII in humans thus has implications for prostate cancer biology and therapeutics.     

Origin of human chromosome 2 revisited

Similarities in chromosome banding patterns and hornologies in DNA sequence between chromosomes of the great apes and humans have suggested that human chromosome 2 originated through the fusion of two ancestral ape chromosomes. A lot of work has been directed at understanding the nature and mechanism of this fusion. The recent availability of the human chrornosome-2-specific alpha satellite DNA probe D2Z and the human chromosome-2p-specific subtelomeric DNA probe D2S445 prompted us to attempt cross-hybridization with chromosomes of the chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla) and orangutan (Pongo pygmaeus) to search for equivalent locations in the great apes and to comment on the origin of human chromosome 2. The probes gave different results. No hybridization to the chromosome-2-specific alpha satellite DNA probe was observed on the presumed homologous great ape chromosomes using both high-stringency and low-stringency post-hybridization washes, whereas the subtelomeric-DNA probe specific for chromosome 2p hybridized to telomeric sites of the short arm of chromosome 12 of all three great apes. These observations suggest an evolutionary difference in the number of alpha satellite DNA repeat units in the equivalent ape chromosomes presumably involved in the chromosome fusion. Nevertheless, complete conservation of DNA sequence of the subtelomeric repeat sequence D2S445 in the ape chromosomes is demonstrated.     

Isolation and comparative mapping of a human chromosome 20-specific alpha-satellite DNA clone.

We have isolated and characterized a human genomic DNA clone (PZ20, locus D20Z2) that identifies, under high-stringency hybridization conditions, an alphoid DNA subset specific for chromosome 20. The specificity was determined using fluorescence in situ hybridization. Sequence analysis confirmed our previously reported data on the great similarity between the chromosome 20 and chromosome 2 alphoid subsets. Comparative mapping of pZ20 on chimpanzee and gorilla chromosomes, also performed under high-stringency conditions, indicates that the alphoid subset has ancestral sequences on chimpanzee chromosome 11 and gorilla chromosome 19. However, no hybridization was observed to chromosomes 21 in the great apes, the homolog of human chromosome 20.     

Reexamination of the African hominoid trichotomy with additional sequences from the primate beta-globin gene cluster.

Additional DNA sequence information from a range of primates, including 13.7 kb from pygmy chimpanzee (Pan paniscus), was added to data sets of beta-globin gene cluster sequence alignments that span the gamma 1, gamma 2, and psi eta loci and their flanking and intergenic regions. This enlarged body of data was used to address the issue of whether the ancestral separations of gorilla, chimpanzee, and human lineages resulted from only one trichotomous branching or from two dichotomous branching events. The degree of divergence, corrected for superimposed substitutions, seen in the beta-globin gene cluster between human alleles is about a third to a half that observed between two species of chimpanzee and about a fourth that between human and chimpanzee. The divergence either between chimpanzee and gorilla or between human and gorilla is slightly greater than that between human and chimpanzee, suggesting that the ancestral separations resulted from two closely spaced dichotomous branchings. Maximum parsimony analysis further strengthened the evidence that humans and chimpanzees share the longest common ancestry. Support for this human-chimpanzee clade is statistically significant at P = 0.002 over a human-gorilla clade or a chimpanzee-gorilla clade. An analysis of expected and observed homoplasy revealed that the number of sequence changes uniquely shared by human and chimpanzee lineages is too large to be attributed to homoplasy. Molecular clock calculations that accommodated lineage variations in rates of molecular evolution yielded hominoid branching times that ranged from 17-19 million years ago (MYA) for the separation of gibbon from the other hominoids to 5-7 MYA for the separation of chimpanzees from humans. Based on the relatively late dates and mounting corroborative evidence from unlinked nuclear genes and mitochondrial DNA for the close sister grouping of humans and chimpanzees, a cladistic classification would place all apes and humans in the same family. Within this family, gibbons would be placed in one subfamily and all other extant hominoids in another subfamily. The later subfamily would be divided into a tribe for orangutans and another tribe for gorillas, chimpanzees, and humans. Finally, gorillas would be placed in one subtribe with chimpanzees and humans in another, although this last division is not as strongly supported as the other divisions.