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     

Potential for retroposition by old Alu subfamilies

Alu elements sharing sequence characteristics of the old subfamilies are thought to currently be retrotranspositionally inactive. We analyzed one of these old subfamilies of Alu elements, Sx, for sequence conservation relative to the consensus and the length of the A-tail as parameters to define the presence of potential Alu Sx source genes in the human genome. Sequence identity to the left half or the right half of the Alu Sx consensus sequence was evaluated for 4424 complete elements obtained from the human genome draft sequence. A small subset of Alu Sx left halves were found to be more conserved than any of the Alu Sx right halves. Selection for promoter function in active elements may explain the slightly higher conservation of the left half. In order to determine whether this sequence identity was the result of recent activity, or simply sequence conservation for older elements, PCR amplification of some of the loci containing Sx elements with conserved left/right halves from different primate genomes was carried out. Several of these Sx Alus were found to have amplified at a later evolutionary period (<35 mya) than expected based on previous studies of Sx elements. Analysis of A-tail length, a feature correlated with current retroposition activity, varied between Alu Sx element loci in different primates, where the length increased in specific Alu elements in the human genome. The presence of few conserved Alu Sx elements and the dynamic expansion/contraction of the A-tail suggests that some of these older subfamilies may still be active at very low levels or in a few individuals. Present address: (Claudina Alemán Stevenson) Laboratory of Cell Biology, NCI/NIH Building 37/Rm 1A09, Bethesda, MD 20892, USA     

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.     

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.     

A human-specific subfamily of Alu sequences.

Of a total of 500,000 Alu family members, approximately 500 are present as a human-specific (HS) subfamily. Each of the HS subfamily members shares a high degree of nucleotide identity and is not present at orthologous positions in other primate genomes, suggesting that HS subfamily members have recently inserted within the human genome. This confirms the hypothesis that the majority of Alu family members are amplified copies of a "master" gene(s). This master gene appears to be amplifying at a rate much slower than that seen earlier in primate evolution. Some of the HS Alu subfamily members have amplified so recently that they are dimorphic in the human population, making them a potentially powerful tool for studies of human populations.     

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.     

Alu repeats and human genomic diversity

During the past 65 million years, Alu elements have propagated to more than one million copies in primate genomes, which has resulted in the generation of a series of Alu subfamilies of different ages. Alu elements affect the genome in several ways, causing insertion mutations, recombination between elements, gene conversion and alterations in gene expression. Alu-insertion polymorphisms are a boon for the study of human population genetics and primate comparative genomics because they are neutral genetic markers of identical descent with known ancestral states.     

A young Alu subfamily amplified independently in human and African great apes lineages.

A variety of Alu subfamilies amplified in primate genomes at different evolutionary time periods. Alu Sb2 belongs to a group of young subfamilies with a characteristic two-nucleotide deletion at positions 65/66. It consists of repeats having a 7-nucleotide duplication of a sequence segment involving positions 246 through 252. The presence of Sb2 inserts was examined in five genomic loci in 120 human DNA samples as well as in DNAs of higher primates. The lack of the insertional polymorphism seen at four human loci and the absence of orthologous inserts in apes indicated that the examined repeats retroposed early in the human lineage, but following the divergence of great apes. On the other hand, similar analysis of the fifth locus (butyrylcholinesterase gene) suggested contemporary retropositional activity of this subfamily. By a semi-quantitative PCR, using a primer pair specific for Sb2 repeats, we estimated their copy number at about 1500 per human haploid genome; the corresponding numbers in chimpanzee and gorilla were two orders of magnitude lower, while in orangutan and gibbon the presence of Sb2 Alu was hardly detectable. Sequence analysis of PCR-amplified Sb2 repeats from human and African great apes is consistent with the model in which the founding of Sb2 subfamily variants occurred independently in chimpanzee, gorilla and human lineages.     

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.     

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.     

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.     

Source gene composition and gene conversion of the AluYh and AluYi lineages of retrotransposons

Background  

Alu elements are a family of SINE retrotransposons in primates. They are classified into subfamilies according to specific diagnostic mutations from the general Alu consensus. It is now believed that there may be several retrotranspositionally-competent source genes within an Alu subfamily. In this study, subfamilies falling on the AluYi and AluYh lineages, and the AluYg6 subfamily, are assessed for the presence of secondary source genes, and the influence of gene conversion on the AluYh and AluYi lineages is also described.     

Alu-SINE exonization: en route to protein-coding function

The majority of more than one million primate-specific Alu elements map to nonfunctional parts of introns or intergenic sequences. Once integrated, they have the potential to become exapted as functional modules, e.g., as protein-coding domains via alternative splicing. This particular process is also termed exonization and increases protein versatility. Here we investigate 153 human chromosomal loci where Alu elements were conceivably exonized. In four selected examples, we generated, with the aid of representatives of all primate infraorders, phylogenetic reconstructions of the evolutionary steps presumably leading to exonization of Alu elements. We observed a variety of possible scenarios in which Alu elements led to novel mRNA splice forms and which, like most evolutionary processes, took different courses in different lineages. Our data show that, once acquired, some exonizations were lost again in some lineages. In general, Alu exonization occurred at various time points over the evolutionary history of primate lineages, and protein-coding potential was acquired either relatively soon after integration or millions of years thereafter. The course of these paths can probably be generalized to the exonization of other elements as well.     

SVA elements: a hominid-specific retroposon family

SVA is a composite repetitive element named after its main components, SINE, VNTR and Alu. We have identified 2762 SVA elements from the human genome draft sequence. Genomic distribution analysis indicates that the SVA elements are enriched in G+C-rich regions but have no preferences for inter- or intragenic regions. A phylogenetic analysis of the elements resulted in the recovery of six subfamilies that were named SVA_A to SVA_F. The composition, age and genomic distribution of the subfamilies have been examined. Subfamily age estimates based upon nucleotide divergence indicate that the expansion of four SVA subfamilies (SVA_A, SVA_B, SVA_C and SVA_D) began before the divergence of human, chimpanzee and gorilla, while subfamilies SVA_E and SVA_F are restricted to the human lineage. A survey of human genomic diversity associated with SVA_E and SVA_F subfamily members showed insertion polymorphism frequencies of 37.5% and 27.6%, respectively. In addition, we examined the amplification dynamics of SVA elements throughout the primate order and traced their origin back to the beginnings of hominid primate evolution, approximately 18 to 25 million years ago. This makes SVA elements the youngest family of retroposons in the primate order.     

Alu element mutation spectra: molecular clocks and the effect of DNA methylation

In primate genomes more than 40% of CpG islands are found within repetitive elements. With more than one million copies in the human genome, the Alu family of retrotransposons represents the most successful short interspersed element (SINE) in primates and CpG dinucleotides make up about 20% of Alu sequences. It is generally thought that CpG dinucleotides mutate approximately ten times faster than other dinucleotides due to cytosine methylation and the subsequent deamination and conversion of C-->T. However, the disparity of Alu subfamily age estimations based upon CpG or non-CpG substitution density indicates a more complex relationship between CpG and non-CpG substitutions within the Alu elements. Here we report an analysis of the mutation patterns for 5296 Alu elements comprising 20 subfamilies. Our results indicate a relatively constant CpG versus non-CpG substitution ratio of approximately 6 for the young (AluY) and intermediate (AluS) Alu subfamilies. However, a more complex non-linear relationship between CpG and non-CpG substitutions was observed when old (AluJ) subfamilies were included in the analysis. These patterns may be the result of the slowdown of the neutral mutation rate during primate evolution and/or an increase in the CpG mutation rate as the consequence of increased DNA methylation in response to a burst of retrotransposition activity approximately 35 million years ago.     

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.