Molecular evolution of intergenic DNA in higher primates: pattern of DNA changes, molecular clock, and evolution of repetitive sequences

A 3.1-kb intergenic DNA fragment located between the psi beta-globin and delta-globin genes in the beta-globin gene cluster was cloned from gorilla, orangutan, rhesus monkey, and spider monkey, and the nucleotide sequence of each fragment was determined. The phylogeny of these four sequences, together with two previously published allelic sequences from humans and one from chimpanzee, was constructed, and the accumulation of mutations in the region was analyzed. The sites of base substitutions are not evenly distributed within the region: two Alu repeats have accumulated 0.21 + 0.02 substitutions/site with 0.15 + 0.008 substitutions/site in the remainder of the fragment. The occurrence of substitutions at neighboring sites is more frequent than would be expected if they were independent. The observed excesses disappear when ancestral -CG- dinucleotide sites are excluded. The phylogenetic relationships of the sequences indicate that the human sequence shares a most recent coancestor with the chimpanzee sequence. The data also show that great apes have accumulated fewer mutations in this part of the genome than has the rhesus monkey. The relative rates of accumulation of 12 kinds of nucleotide substitution in the region during primate evolution are asymmetric in the DNA strands. From these rates of accumulation, the origin of a simple stretch of sequence near the 3' end of the 3.1-kb fragment was deduced to be a sequence comprising 50% T and 50% C on one strand. The two oppositely oriented Alu sequences in the 3.1-kb region were inserted at their present positions before the divergence of the New-World monkeys from other lineages. Our analysis shows that the nucleotide sequences of the two Alu repeats in spider monkey are unexpectedly similar both to each other and to the deduced ancestral sequence of Alu repeats. The data suggest that there has been some type of recombinational event between the spider monkey Alu repeats but that it was not a simple gene conversion.      

The beta globin gene cluster of the prosimian primate Galago crassicaudatus: nucleotide sequence determination of the 41-kb cluster and comparative sequence analyses.

The nucleotide sequence of the beta globin gene cluster of the prosimian Galago crassicaudatus has been determined. A total sequence spanning 41,101 bp contains and links together previously published sequences of the five galago beta-like globin genes (5'-epsilon-gamma-psi eta-delta-beta-3'). A computer-aided search for middle interspersed repetitive sequences identified 10 LINE (L1) elements, including a 5' truncated repeat that is orthologous to the full-length L1 element found in the human epsilon-gamma intergenic region. SINE elements that were identified included one Alu type I repeat, four Alu type II repeats, and two methionine tRNA-derived Monomer (type III) elements. Alu type II and Monomer sequences are unique to the galago genome. Structural analyses of the cluster sequence reveals that it is relatively A+T rich (about 62%) and regions with high G+C content are associated primarily with globin coding regions. Comparative analyses with the beta globin cluster sequences of human, rabbit, and mouse reveal extensive sequence homologies in their genic regions, but only human, galago, and rabbit sequences share extensive intergenic sequence homologies. Divergence analyses of aligned intergenic and flanking sequences from orthologous human, galago, and rabbit sequences show a gradation in the rate of nucleotide sequence evolution along the cluster where sequences 5' of the epsilon globin gene region show the least sequence divergence and sequences just 5' of the beta globin gene region show the greatest sequence divergence.     

Allelic dimorphism in the human tissue-type plasminogen activator (TPA) gene as a result of an Alu insertion/deletion event

Summary Polymerase chain reaction and direct sequencing were used to investigate an amplified DNA fragment containing the suspected polymorphic site of all known intragenic restriction fragment length polymorphisms (RFLPs) within the human tissue-type plasminogen activator (TPA) gene. Sequence data obtained showed that these RFLPs were all generated by the presence or absence of one of the two Alu sequences located in intron h of the human TPA gene. Furthermore, one of the direct repeats flanking this Alu sequence was absent in the minor allele. In addition to indicating the presence of an Alu insertion in an ancestral human TPA gene, these findings suggest a slip-replication mechanism for the deletion of this Alu repeat, once inserted into the gene. As both alleles have been observed in similar frequencies among different ethnic groups, the insertion or subsequent deletion of this Alu sequence in the human TPA gene must have occurred early in human evolution.     

Evolution of alu family repeats since the divergence of human and chimpanzee

Summary The DNA sequences of three members of the Alu family of repeated sequences located 5 to the chimpanzee 2 gene have been determined. The base sequences of the three corresponding human Alu family repeats have been previously determined, permitting the comparison of identical Alu family members in human and chimpanzee. Here we compare the sequences of seven pairs of chimpanzee and human Alu repeats. In each case, with the exception of minor sequence differences, the identical Alu repeat is located at identical sites in the human and chimpanzee genomes. The Alu repeats diverge at the rate expected for nonselected sequences. Sequence conversion has not replaced any of these 14 Alu family members since the divergence between chimpanzee and human.     

Primate evolution of the alpha-globin gene cluster and its Alu-like repeats

The arrangement of alpha-globin genes in Old World and New World monkeys and a prosimian, galago, has been determined by restriction mapping. Recombinant DNAs containing galago and Old World monkey alpha-globin genes have been isolated and subjected to a partial sequence determination for comparison to alpha-globin genes in human, chimpanzee and non-primate mammals. The results of this extensive structural analysis are relevant to several topics concerning the evolution of primate alpha-globin genes and Alu family repeats. All orders of higher primates (i.e. Old and New World monkeys, chimpanzee and human) have the same arrangement of alpha-globin genes. In contrast, the arrangement and correction of galago alpha-globin genes differ from those of higher primates, but are similar to those of non-primate mammals. The 5' and 3'-flanking regions of the human alpha 1 gene are orthologous to the corresponding region in galago, identifying the human alpha 2 gene as the more recently duplicated gene. The human psi alpha 1 gene is found to be inactivated after divergence of the human and galago lineages but prior to the divergence of human and monkey. Orthologous Alu family members in human and monkey DNAs indicate that the dispersion of some Alu repeats occurred prior to the divergence of these lineages. However, the Alu-like repeats of prosimian and higher primates result from entirely independent events giving rise to different repeat elements inserted at distinct genomic positions.     

Structure of the gorilla alpha-fetoprotein gene and the divergence of primates.

The sequence of the gorilla alpha-fetoprotein gene, including 869 base pairs of the 5' flanking region and 4892 base pairs of the 3' flanking region (24,607 in total), was determined from two overlapping lambda phage clones. The sequence extends 18,846 base pairs from the Cap site to the polyadenylation site, and it reveals that the gene is composed of 15 exons, which are symmetrically placed within three domains of alpha-fetoprotein. The deduced polypeptide chain is composed of a 19-amino-acid leader peptide, followed by 590 amino acids of the mature protein. The RNA polymerase II binding site, TATAAAA, and the promoter element, CCAAC, are positioned at -21 and -65 from the Cap site, respectively. The polyadenylation signal, AATAAA, is located in the last exon, which is untranslated. The sequence for the gorilla alpha-fetoprotein gene was compared with that of the previously published human alpha-fetoprotein gene (P. E. M. Gibbs, R. Zielinski, C. Boyd, and A. Dugaiczyk, 1987, Biochemistry 26: 1332-1343). Four types of repetitive sequence elements were found in identical positions in both species. However, one Alu and one Xba DNA repeat within introns 4 and 7, respectively, of the human gene are absent from orthologous positions in the gorilla. The Alu and the Xba DNA repeats probably emerged in the human genome after the human/gorilla divergence and became established novelties in the human lineage. There are 363/21,523 mutational changes between human and gorilla, amounting to 1.69% DNA divergence between the two primate species. The value of 1.69% is lower than the 2.27% obtained from melting temperatures of hybrids between human and gorilla genomic DNA (C. G. Sibley and J. E. Ahlquist, 1984, J. Mol. Evol. 26: 99-121). At the protein level, Homo sapiens differs from Gorilla gorilla only at 4 of 609 amino acid positions (0.66%) in the alpha-fetoprotein sequence. This difference signifies a lower rate of molecular divergence for the alpha-fetoprotein gene in primates, as compared to rodents.     

The human apolipoprotein AII gene: structural organization and sites of expression.

The complete nucleotide sequence of the human apolipoprotein All gene together with 911 bases of 5' flanking sequence and 687 bases of 3' flanking sequence have been determined. The mRNA coding region is interrupted by three introns of 169, 293 and 395bp. The Intro-exon structure of the apo All gene is similar to that of the apo AI, apo CIII and apo E genes: three introns separate 4 coding sequences specifying the 5' untranslated region, pre-peptide, a short N-terminal domain and a C-terminal domain composed of a variable number of lipid-binding amphipathic helices. Intron II carries a 33bp dG-dT repetitive element adjacent to the 3' splice junction which has the potential to adopt the Z-DNA conformation. The 5' and 3' terminuses of the mRNA have been identified by primer extension and S1 nuclease mapping. A number of short direct repeats are found in the 5' flanking region and an inverted repeat occurs between the CAAT and TATA boxes. Downstream of the the gene is an Alu family repeat containing a polymorphic MspI site, the deletion of which is associated with increased circulating levels of apoAII. ApoAII gene expression was demonstrated in adult human liver and HepG2 cells but not in human small intestine. Of ten Rhesus monkey tissues examined apo All mRNA was detected only in liver.     

Various aspects of evolution of Alu repeats in mammals

A complex study on various evolutionary peculiarities of the mammalia dispersed Alu repeats (Alu repeats of primates and B1 of rodents) has been carried out by phylogenetic analysis. A phylogenetic tree, containing the 7SL RNA genes and the Alu repeats of primates and rodents has been constructed. It has been shown that the branch of the phyletic line leading to the Alu repeats of primates and B1 of rodents from the 7SL RNA genes occurred after the divergence of the 7SL RNA genes of amphibia and mammalia, but before the divergence of the 7SL RNA genes of primates and rodents (250.10 years ago). A statistically reliable slowing down in the evolutionary rates of one of two monomers for the human Alu repeats has been proved. It may be caused by the functional load of the corresponding monomer in connection with the presence of a definit regulatory site in it.     

Two human gamma-crystallin genes are linked and riddled with Alu-repeats

A human genomic cosmid clone, pHcos gamma-1, has been isolated containing two closely linked gamma-crystallin genes, oriented in the same direction. The sequence of these genes and their 5' and 3' flanking regions has been determined. The coding regions of both genes are interrupted by two introns. The first introns (94 and 100 bp, respectively) are located in the 5' region of the genes. The second introns (2.82 and 0.95 kb, respectively) divide the genes into two halves, each encoding a structural domain of the gamma-crystallin protein. The coding regions of the two genes show 80% homology. Due to a mutation in the splice acceptor site of the second intron of the first gene, the coding region of its third exon is 3 bp longer than that of the second gene. In the flanking regions several conserved sequence elements were found, including those elements that are known to be necessary for the correct expression of eukaryotic genes. The flanking and intronic regions of the genes contain 'simple sequence' DNA and Alu repeats. The Alu repeats are usually clustered, contain truncated elements, and are often located near simple sequence DNA.     

Invasion of the human albumin-alpha-fetoprotein gene family by Alu, Kpn, and two novel repetitive DNA elements

The human albumin-alpha-fetoprotein genomic domain contains 13 repetitive DNA elements randomly distributed throughout the symmetrical structures of these genes. These repeated sequences are located at different sites within the two genes. The human albumin gene contains five Alu elements within four of its 14 intervening sequences. Two of these repeats are located in intron 2, and the remaining three are located in introns 7, 8, and 11. The human alpha-fetoprotein gene contains three of these Alu elements, one in intron 4 and the remaining two in the 3'-untranslated region. In addition, the human alpha-fetoprotein gene contains a Kpn repeat and two classes of novel repeats that are absent from the human albumin gene. Six of the Alu elements within the two genes are bound by short direct repeats that harbor five base substitutions in 120 possible positions (60 bp times 2 termini). The absence of Alu repeats from analogous positions in rodents indicates that these repeats invaded the albumin-alpha-fetoprotein domain less than 85 Myr ago (the time of mammalian radiation). Furthermore, considering the conservation of terminal repeats flanking the Alu sequences of the albumin-alpha-fetoprotein domain (0.042 changes per site), we submit that the average time of Alu insertion into this gene family could have been as recently as 15-30 Myr ago.     

A human dimorphism resulting from loss of an Alu.

The molecular phylogeny of Alu and other repeated sequences in the human genome provides clues to events during primate evolution. A subclass of human Alu's has been previously identified as dimorphic insertions within members of the medium reiteration frequency (mer) class of repeats, reflecting the complicated sequence of insertion and radiation events leading to the current human genome structure. One dimorphic Alu is located within a previously unidentified mer family member, in the first intron of the human T4 (CD4) gene. The insertion (Alu+ allele) has a frequency of approximately 70% in Europeans and Africans and is homozygous in 20 Asian samples. Polymerase chain reaction amplification, direct DNA sequencing, and Southern analysis using oligonucleotide probes revealed that the Alu- allele was derived from the Alu+ allele by loss of part of the inserted sequence. Comparison with a tightly linked marker within the human genome and studies of baboon DNA samples revealed that the original insertion was a relatively early event in primate evolution, but that the Alu sequence loss leading to the dimorphism has occurred much more recently. Loss of Alu insertions therefore represents one mechanism for the generation of human Alu dimorphisms.     

Recombinational biases in the rearranged C1-inhibitor genes of hereditary angioedema patients

DNA structural changes responsible for hereditary angioedema were sought in the C1-inhibitor gene, which contains unusually dense clusters of Alu repeats in various orientations. Among patients belonging to 45 unrelated families, eight partial C1-inhibitor gene deletions and a partial duplication were found. Four deletions had one of the boundaries within the gene and the other in extragenic regions--in three cases 5' of the gene and in one case 3' of the gene. The boundaries of the partial duplication and of the remaining four deletions mapped instead within a few kilobases of exon 4. The same element--Alu 1--the first of three tandem Alu repeats preceding exon 4, contained one of the breakpoints of each of these five rearrangements. Moreover, these recombination breakpoints spread over the entire length of Alu 1, in contrast with the tight clustering observed near the 5' end of Alu sequences rearranged in other human genes. Thus, two uncommon recombinational biases are observed in the Alu rearrangements of hereditary angioedema patients; one promotes the occurrence of intragenic breakpoints in a single Alu repeat, and the other allows the breaks to be distributed over the entire Alu structure rather than within the hot spot of the left Alu monomer. A region of potential Z-DNA structure, located 1.7 kb upstream of Alu 1, may contribute to both peculiarities.     

Analysis of the regions flanking the human insulin gene and sequence of an Alu family member.

The regions around the human insulin gene have been studied by heteroduplex, hybridization and sequence analysis. These studies indicated that there is a region of heterogeneous length located approximately 700 bp before the 5' end of the gene; and that the 19 kb of cloned DNA which includes the 1430 bp insulin gene as well as 5650 bp before and 11,500 bp after the gene is single copy sequence except for 500 bp located 6000 bp from the 3' end of the gene. This 500 bp segment contains a member of the Alu family of dispersed middle repetitive sequences as well as another less highly repeated homopolymeric segment. The sequence of this region was determined. This Alu repeat is bordered by 19 bp direct repeats and also contains an 83 bp sequence which is present twice. The regions flanking the human and rat I insulin genes were compared by heteroduplex analysis to localize homologous sequences in the flanking regions which could be involved in the regulation of insulin biosynthesis. The homology between the two genes is restricted to the region encoding preproinsulin and a short region of approximately 60 bp flanking the 5' side of the genes.     

Alpha-galactosidase A gene rearrangements causing Fabry disease. Identification of short direct repeats at breakpoints in an Alu-rich gene

Fabry disease, an inborn error of glycosphingolipid catabolism, results from mutations in the X-linked gene encoding the lysosomal enzyme, alpha-galactosidase A (EC 3.2.1.22). Six alpha-galactosidase A gene rearrangements that cause Fabry disease were investigated to assess the role of Alu repetitive elements and short direct and/or inverted repeats in the generation of these germinal mutations. The breakpoints of five partial gene deletions and one partial gene duplication were determined by either cloning and sequencing the mutant gene from an affected hemizygote, or by polymerase chain reaction amplifying and sequencing the genomic region containing the novel junction. Although the alpha-galactosidase A gene contains 12 Alu repetitive elements (representing approximately 30% of the 12-kilobase (kb) gene or approximately 1 Alu/1.0 kb), only one deletion resulted from an Alu-Alu recombination. The remaining five rearrangements involved illegitimate recombinational events between short direct repeats of 2 to 6 base pairs (bp) at the deletion or duplication breakpoints. Of these rearrangements, one had a 3' short direct repeat within an Alu element, while another was unusual having two deletions of 1.7 kb and 14 bp separated by a 151-bp inverted sequence. These findings suggested that slipped mispairing or intrachromosomal exchanges involving short direct repeats were responsible for the generation of most of these gene rearrangements. There were no inverted repeat sequences or alternating purine-pyrimidine regions which may have predisposed the gene to these rearrangements. Intriguingly, the tetranucleotide CCAG and the trinucleotide CAG (or their respective complements, CTGG and CTG) occurred within or adjacent to the direct repeats at the 5' breakpoints in three and four of the five alpha-galactosidase A gene rearrangements, respectively, suggesting a possible functional role in these illegitimate recombinational events. These studies indicate that short direct repeats are important in the formation of gene rearrangements, even in human genes like alpha-galactosidase A that are rich in Alu repetitive elements.     

PROGINS Alu insertion and human genomic diversity

A polymorphic Alu element belonging to the young Ya5 subfamily of Alu repeats located in the progesterone receptor gene has been characterized. Using a polymerase chain reaction (PCR)-based assay, the genetic diversity associated with the PROGINS Alu repeat was determined in a diverse array of human populations. The level of insertion polymorphism associated with PROGINS suggests that it will be a useful marker for the study of human evolution. In addition, we determined the distribution of the PROGINS Alu insertion in two groups of women from greater New Orleans, LA with breast cancer. The PROGINS Alu insertion was not associated with breast cancer in the populations tested.     

Existence of at least three distinct Alu subfamilies

Summary Computer-assisted sequence analysis of human Alu family members reveals that Alu repeats belong to one of at least three subfamilies. The insertion of human Alu repeats can be represented by three episodic bursts, each of which was founded by a distinct master sequence.     

An unusual Alu repeat sequence within the CAD gene

Summary There are several hundred thousand members of the Alu repeat family in the human genome. Those Alu elements sequenced to date appear to fit into subfamilies. A novel Alu has been found in an intron of the human CAD gene: it appears to be due to rearrangement between Alu repeats belonging to two different subfamilies. Further sequence data from this intron suggest that the Alu element may have rearranged prior to its entry into the CAD gene. Such findings indicate that, in addition to single nucleotide substitutions and deletions, DNA rearrangments may be a factor in generating the diversity of Alu repeats found in primate genomes.