Evolution of the master Alu gene(s)

Summary A comparison of Alu sequences that comprise more recently amplified Alu subfamilies was made. There are 18 individual diagnostic mutations associated with the different subfamilies. This analysis confirmed that the formation of each subfamily can be explained by the sequential accumulation of mutations relative to the previous subfamily. Polymerase chain reaction amplification of orthologous loci in several primate species allowed us to determine the time of insertion of Alu sequences in individual loci. These data suggest that the vast majority of Alu elements amplified at any given time comprised a single Alu subfamily. We find that, although the individual divergence relative to a consensus sequence correlate reasonably well with sequence age, the diagnostic mutations are a more accurate measure of the age of any individual Alu family member. Our data are consistent with a model in which all Alu family members have been made from a single master gene or from a series of sequential master genes. This master gene(s) accumulated diagnostic base changes, resulting in the amplification of different subfamilies from the master gene at different times in primate evolution. The changes in the master gene(s) probably occurred individually, but their appearance is clearly punctuated. Ten of them have occurred within an 15-million-year time span, 40–25 million years ago, and 8 changes have occurred within the last 5 million years. Surprisingly, no changes appeared in the 20 milion years separating these periods.     

Recently integrated human Alu repeats: finding needles in the haystack

Alu elements undergo amplification through retroposition and integration into new locations throughout primate genomes. Over 500,000 Alu elements reside in the human genome, making the identification of newly inserted Alu repeats the genomic equivalent of finding needles in the haystack. Here, we present two complementary methods for rapid detection of newly integrated Alu elements. In the first approach we employ computational biology to mine the human genomic DNA sequence databases in order to identify recently integrated Alu elements. The second method is based on an anchor-PCR technique which we term Allele-Specific Alu PCR (ASAP). In this approach, Alu elements are selectively amplified from anchored DNA generating a display or 'fingerprint' of recently integrated Alu elements. Alu insertion polymorphisms are then detected by comparison of the DNA fingerprints generated from different samples. Here, we explore the utility of these methods by applying them to the identification of members of the smallest previously identified subfamily of Alu repeats in the human genome termed Ya8. This subfamily of Alu repeats is composed of about 50 elements within the human genome. Approximately 50% of the Ya8 Alu family members have inserted in the human genome so recently that they are polymorphic, making them useful markers for the study of human evolution. This revised version was published online in July 2006 with corrections to the Cover Date.     

Structure and variability of recently inserted Alu family members.

The HS subfamily of Alu sequences is comprised of a group of nearly identical members. Individual subfamily members share 97.7% nucleotide identity with each other and 98.9% nucleotide identity with the HS consensus sequence. Individual subfamily members are on the average 2.8 million years old, and were probably derived from a single source 'master' gene sometime after the human/great ape divergence. The recent Alu family member insertions provide a better image of the structure of Alu retroposons before they have had the opportunity to change significantly. All of the HS subfamily members are flanked by perfect direct repeats as a result of insertion at staggered nicks. The 'master' gene from which the HS subfamily members were derived had an oligo-dA rich tail at least 40 bases long. The 'master' gene is very rich in CpG dinucleotides, but nucleotide substitutions within subfamily members accumulated in a random manner typical for Alu sequence with CpG substitutions occurring 9.2 fold faster than non-CpG substitutions.     

The role and amplification of the HS Alu subfamily founder gene

A recently identified Alu element (Leeflang et al. J. Mol. Evol. 1993, 37:559–565), referred to as the putative founder of the HS (PV) subfamily, was found to be present at orthologous loci in the human, chimpanzee, gorilla, and gibbon lineages. The evolution of this Alu suggested that it is a source gene in the evolution of Alu family repeats for one of the most recent subfamilies, HS. We have determined that this putative founder of the HS subfamily was not present at the orthologous loci in older primates, including old world and new world monkeys. Thus, this particular Alu locus has only been responsible for the establishment of a very small subfamily of Alu sequences. We have further demonstrated that this putative founder Alu was not responsible for the de novo Alu insertion into the neurofibromatosis-1 gene of an individual causing neurofibromatosis. Our data demonstrate that although the putative founder of the HS subfamily found by Leeflang et al. (1993) probably gave rise to one of the most recent subfamilies of Alu sequences, it has not been very active in retroposition. Correspondence to: T.H. Shaikh     

Comprehensive analysis of two Alu Yd subfamilies

Alu elements have inserted in the human genome throughout primate evolution. A small number of Alu insertions have occurred after the divergence of humans from nonhuman primates and therefore should not be present in nonhuman primate genomes. Most of these recently integrated Alu elements are contained with a series of discrete Alu subfamilies that are related to each other based upon diagnostic nucleotide substitutions. We have extracted members of the Alu Yd subfamily that are derivatives of the Alu Y subfamily that share a common 12-bp deletion that defines the Yd lineage from the draft sequence of the human genome. Analysis of the Yd Alu elements resulted in the recovery of two new Alu subfamilies, Yd3 and Yd6, which contain a total of 295 members (198 Yd3 and 97 Yd6). DNA sequence analysis of each of the Alu Yd subfamilies yielded age estimates of 8.02 and 1.20 million years old for the Alu Yd3 and Yd6 subfamilies, respectively. Two hundred Alu Yd3 and Yd6 loci were screened using polymerase chain reaction (PCR) assays to determine their phylogenetic origin and associated levels of human genomic diversity. The Alu Yd3 subfamily appears to have started amplifying relatively early in primate evolution and continued propagating albeit at a low level as many of its members are found in a variety of hominoid (humans, greater and lesser ape) genomes. Only two of the elements are polymorphic in the human genome and absent from the genomes of nonhuman primates. By contrast all of the members of the Alu Yd6 subfamily are restricted to the human genome, with 12% of the elements representing insertion polymorphisms in human populations. A single Alu Yd6 locus contained an independent parallel forward insertion of a paralogous Alu Sq sequence in the owl monkey. These Alu subfamilies are a source of genomic fossil relics for the study of primate phylogenetics and human population genetics.     

Amplification dynamics of human-specific (HS) Alu family members.

We have investigated the distribution of several recently inserted Alu family members within representatives of diverse human groups. Human population studies using 65 unrelated human DNA samples, as well as a familial study to test inheritance, showed that individual Alu family members could be divided into three groups. The first group consisted of relatively older Alu family members which were monomorphic (homozygous) throughout the population tested (HS C3N1 and C4N6). The second group (HS C4N2, C4N5 and C4N8), apparently inserted into other repetitive regions of the genome, resulting in inconclusive results in the PCR test used. However, it is clear that these particular Alu insertions were present in a majority if not all of the loci tested. The third group was comprised of three dimorphic Alu family members (HS C2N4, C4N4 and TPA 25). Only a single Alu family member (TPA 25) displayed a high degree of dimorphism within the human population. This latter example also showed different allele frequencies in different human groups. The isolation and characterization of additional highly dimorphic Alu family members should provide a useful tool for human population genetics.     

The Alu Yc1 subfamily: sorting the wheat from the chaff

Members of the Alu Yc1 subfamily are distinguished from the older Alu Y subfamily by a signature G-->A substitution at base 148 of their 281-bp consensus sequence. Members of the much older and larger Alu Y subfamily could have by chance accumulated this signature G-->A substitution and be misclassified as belonging to the Alu Yc1 subfamily. Using a Mahanalobis classification method, it was estimated that the "authentic" Alu Yc1 subfamily consists of approximately 262 members in the human genome. PCR amplification and further analysis was successfully completed on 225 of the Yc1 Alu family members. One hundred and seventy-seven Yc1 Alu elements were determined to be monomorphic (fixed for presence) in a panel of diverse human genomes. Forty-eight of the Yc1 Alu elements were polymorphic for insertion presence/absence in diverse human genomes. The insertion polymorphism rate of 21% in the human genome is similar to rates reported previously for other "young" Alu subfamilies. The polymorphic Yc1 Alu elements will be useful genetic loci for the study of human population genetics.     

Clustering and subfamily relationships of the Alu family in the human genome

Thirteen and 10 sequences of the Alu family of repeated DNA elements found within the human thymidine kinase and beta-tubulin genes, respectively, were compared. These genes have approximately five times the expected density of Alu family members. The consensus sequence that could be drawn from these 23 Alu family members would differ slightly from others drawn from random Alu family sequences but only at very heterogeneous positions. The different Alu family members do show different pairwise percentage identities, with approximately 15% (7 of 48 Alu family members analyzed) of them clearly representing a separate subfamily of sequences. This analysis also confirms the species- specific differences between human and the prosimian Galago crassicaudatus Alu family members. These data are consistent with both the origin of these sequences in primates less than 65-70 Myr ago and amplification since that time to their present 500,000 copies. The data do not show any special relationships among densely clustered Alu family members.      

Gene conversion as a secondary mechanism of short interspersed element (SINE) evolution.

The Alu repetitive family of short interspersed elements (SINEs) in primates can be subdivided into distinct subfamilies by specific diagnostic nucleotide changes. The older subfamilies are generally very abundant, while the younger subfamilies have fewer copies. Some of the youngest Alu elements are absent in the orthologous loci of nonhuman primates, indicative of recent retroposition events, the primary mode of SINE evolution. PCR analysis of one young Alu subfamily (Sb2) member found in the low-density lipoprotein receptor gene apparently revealed the presence of this element in the green monkey, orangutan, gorilla, and chimpanzee genomes, as well as the human genome. However, sequence analysis of these genomes revealed a highly mutated, older, primate-specific Alu element was present at this position in the nonhuman primates. Comparison of the flanking DNA sequences upstream of this Alu insertion corresponded to evolution expected for standard primate phylogeny, but comparison of the Alu repeat sequences revealed that the human element departed from this phylogeny. The change in the human sequence apparently occurred by a gene conversion event only within the Alu element itself, converting it from one of the oldest to one of the youngest Alu subfamilies. Although gene conversions of Alu elements are clearly very rare, this finding shows that such events can occur and contribute to specific cases of SINE subfamily evolution.     

Analysis of the human Alu Ya-lineage

The Alu Ya-lineage is a group of related, short interspersed elements (SINEs) found in primates. This lineage includes subfamilies Ya1-Ya5, Ya5a2 and others. Some of these subfamilies are still actively mobilizing in the human genome. We have analyzed 2482 elements that reside in the human genome draft sequence and focused our analyses on the 2318 human autosomal Ya Alu elements. A total of 1470 autosomal loci were subjected to polymerase chain reaction (PCR)-based assays that allow analysis of individual Ya-lineage Alu elements. About 22% (313/1452) of the Ya-lineage Alu elements were polymorphic for the insertion presence on human autosomes. Less than 0.01% (5/1452) of the Ya-lineage loci analyzed displayed insertions in orthologous loci in non-human primate genomes. DNA sequence analysis of the orthologous inserts showed that the orthologous loci contained older pre-existing Y, Sc or Sq Alu subfamily elements that were the result of parallel forward insertions or involved in gene conversion events in the human lineage. This study is the largest analysis of a group of "young", evolutionarily related human subfamilies. The size, evolutionary age and variable allele insertion frequencies of several of these subfamilies makes members of the Ya-lineage useful tools for human population studies and primate phylogenetics.     

Non-traditional Alu evolution and primate genomic diversity

Alu elements belonging to the previously identified "young" subfamilies are thought to have inserted in the human genome after the divergence of humans from non-human primates and therefore should not be present in non-human primate genomes. Polymerase chain reaction (PCR) based screening of over 500 Alu insertion loci resulted in the recovery of a few "young" Alu elements that also resided at orthologous positions in non-human primate genomes. Sequence analysis demonstrated these "young" Alu insertions represented gene conversion events of pre-existing ancient Alu elements or independent parallel insertions of older Alu elements in the same genomic region. The level of gene conversion between Alu elements suggests that it may have a significant influence on the single nucleotide diversity within the genome. All the instances of multiple independent Alu insertions within the same small genomic regions were recovered from the owl monkey genome, indicating a higher Alu amplification rate in owl monkeys relative to many other primates. This study suggests that the majority of Alu insertions in primate genomes are the products of unique evolutionary events.     

Human-specific subfamilies of HERV-K (HML-2) long terminal repeats: three master genes were active simultaneously during branching of hominoid lineages

Using 40 known human-specific LTR sequences, we have derived a consensus sequence for an evolutionary young HERV-K (HML-2) LTR family, which was named the HS family. In the human genome the HS family is represented by approximately 150-160 LTR sequences, 90% of them being human-specific (hs). The family can be subdivided into two subfamilies differing in five linked nucleotide substitutions: HS-a and HS-b of 5.8 and 10.3 Myr evolutionary ages, respectively. The HS-b subfamily members were transpositionally active both before the divergence of the human and chimpanzee ancestor lineages and after it in both lineages. The HS-a subfamily comprises only hs LTRs. These and other data strongly suggest that at least three "master genes" of HERV-K (HML-2) LTRs were active in the human ancestor lineage after the human-chimpanzee divergence. We also found hs HERV-K (HML-2) LTRs integrations in introns of 12 human genes and identified 13 new hs HERV-K (HML-2) LTRs.     

Genetic variation of recent Alu insertions in human populations

The Alu family of intersperesed repeats is comprised of ovr 500,000 members which may be divided into discrete subfamilies based upon mutations held in common between members. Distinct subfamilies of Alu sequences have amplified within the human genome in recent evolutionary history. Several individual Alu family members have amplified so recently in human evolution that they are variable as to presence and absence at specific loci within different human populations. Here, we report on the distribution of six polymorphic Alu insetions in a survey of 563 individuals from 14 human population groups across several continents. Our results indicate that these polymorphic Alu insertions probably have an African origin and that there is a much smaller amount of genetic variation between European populations than that found between other populations groups. Present address: Department of Pathology, Stanley S. Scott Cancer Center, Louisiana State University Medical Center, 1901 Perdido St., New Orleans, LA 70112 Correspondence to: M.A. Batzer     

Mobility of short interspersed repeats within the chimpanzee lineage

The PV subfamily of Alu repeats in human DNA is largely composed of recently inserted members. Here we document additional members of the PV subfamily that are found in chimpanzee but not in the orthologous loci of human and gorilla, confirming the relatively recent and independent expansion of this Alu subfamily in the chimpanzee lineage. As further evidence for the youth of this Alu subfamily, one PV Alu repeat is specific to Pan troglodytes, whereas others are present in Pan paniscus as well. The A-rich tails of these Alu repeats have different lengths in Pan paniscus and Pan troglodytes. The dimorphisms caused by the presence and absence of PV Alu repeats and the length polymorphisms attributed to their A-rich tails should provide valuable genetic markers for molecular-based studies of chimpanzee relationships. The existence of lineage-specific Alu repeats is a major sequence difference between human and chimpanzee DNAs. Correspondence to: C.W. Schmid     

Recently amplified Alu family members share a common parental Alu sequence.

Three of the most recently inserted primate Alu family members are exceptionally closely related. Therefore, one, or a few, Alu family members are dominating the amplification process and the vast majority are not actively involved in retroposition. Although individual Alu family members are not under any apparent evolutionary constraint, the sequences of these active members are being moderately conserved.     

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.     

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.     

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.