The age of Alu subfamilies

Using Kimura's distance measure we have calculated the average age of all major Alu subfamilies based on the most recent available data. We conclude that AluJ sequences are some 26 Myr older than previously thought. Furthermore, the origin of the FLA (Free Left Arm) Alu family can be traced back to the very beginning of the mammalian radiation.One new minor subfamily is reported and discussed in the context of sequence diversity in major Alu subfamilies. Correspondence to: J. Jurka     

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.     

Sequence correlation between neighboring Alu instances suggests post-retrotransposition sequence exchange due to Alu gene conversion

Alu elements constitute 10% of the human genome. Alu mobilization is important in the evolution of the human genome. While retrotransposition is the primary pathway of Alu mobilization, Alu gene conversion has been postulated as a secondary pathway for Alu mobilization in human genome. However, the mode and tempo of Alu gene conversions remain a mystery due to lack of sensitive statistical methods. In this paper, we present the first study on sequence correlation between Alu instances, measured by the number of shared mutations away from the Alu consensus, or co-mutations. Our analysis reveals a significantly elevated co-mutation rate between Alu instances that are located in close proximity along a chromosome. This effect is more pronounced outside Alu subfamily diagnostic positions. This effect peaks among immediately adjacent Alu instances, diminishes quickly in increasing distances between Alu instances, and vanishes beyond 5000 bp. Our results suggest that this effect reflects post-retrotransposition sequence exchanges between Alu instances, mainly due to Alu gene conversions.     

Most human Alu and murine B1 repeats are unique

Alus and B1s are short interspersed repeat elements (SINEs) indirectly derived from the 7SL RNA gene. While most researchers recognize that there exists extensive variability between individual elements, the extent of this variability has never been systematically tested. We examined all Alu elements over 200 nucleotides and all B1 elements over 100 nucleotides in the human and mouse genomes, and analyzed the number of copies of each element at various stringencies from 22 nucleotides to full length. Over 98% of 923,277 Alus and 365,377 B1s examined were unique when queried at full length. When the criterion was reduced to half the length of the repeat, 97% of the Alus and 73% of the B1s were still found to be a single copy. All single and multi-copy sequences have been mapped and documented. Access to the data is possible using the AluPlus website http://www.ibr.hawaii.edu.     

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.     

Alu insertion polymorphisms and an assessment of the genetic contribution of Central Asia to Anatolia with respect to the Balkans

In the evolutionary history of modern humans, Anatolia acted as a bridge between the Caucasus, the Near East, and Europe. Because of its geographical location, Anatolia was subject to migrations from multiple different regions throughout time. The last, well-known migration was the movement of Turkic speaking, nomadic groups from Central Asia. They invaded Anatolia and then the language of the region was gradually replaced by the Turkic language. In the present study, insertion frequencies of 10 Alu loci (A25 = 0.07, APO = 0.96, TPA25 = 0.44, ACE = 0.37, B65 = 0.57, PV92 = 0.18, FXIIIB = 0.52, D1 = 0.40, HS4.32 = 0.66, and HS4.69 = 0.30) have been determined in the Anatolian population. Together with the data compiled from other databases, the similarity of the Anatolian population to that of the Balkans and Central Asia has been visualized by multidimensional scaling method. Analysis suggested that, genetically, Anatolia is more closely related with the Balkan populations than to the Central Asian populations. Central Asian contribution to Anatolia with respect to the Balkans was quantified with an admixture analysis. Furthermore, the association between the Central Asian contribution and the language replacement episode was examined by comparative analysis of the Central Asian contribution to Anatolia, Azerbaijan (another Turkic speaking country) and their neighbors. In the present study, the Central Asian contribution to Anatolia was estimated as 13%. This was the lowest value among the populations analyzed. This observation may be explained by Anatolia having the lowest migrant/resident ratio at the time of migrations.     

An Alu insertion polymorphism in a baboon hybrid zone

A novel polymerase chain reaction (PCR) primer pair was used to analyze the frequency of insertion of the first described, nonhuman, baboon-specific Alu repetitive element in populations from the Papio hamadryas anubis and the Papio hamadryas hamadryas subspecies, and from a number of anubis-hamadryas hybrids. The Alu insertion is found in intron 7 of the baboon lipoprotein lipase (LPL) gene. Each of the populations had different frequencies for the insertion, and the hybrids examined had a frequency intermediate to that of the parental populations. All hybrids and all P. h. anubis groups except the group of anubis sampled in 1973 exhibited higher-than-expected heterozygosity, while P. h. hamadryas and 1973 P. h. anubis showed lower-than-expected heterozygosity, supporting behavioral and other genetic observations of greater anubis outbreeding relative to hamadryas. This may include asymmetric introgression of the Alu insertion from hamadryas to the anubis population due to hybridization. Am J Phys Anthropol 109:1–8, 1999. © 1999 Wiley-Liss, Inc.     

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.     

Alu DNA polymorphism in ACE gene is protective for age-related macular degeneration

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. We report an association between an Alu polymorphism in the angiotensin-converting enzyme (ACE) gene with the dry/atrophic form of AMD. Using the polymerase chain reaction (PCR) on genomic DNA isolated from patients with AMD (n=173), and an age-matched control population (n=189), we amplified a region polymorphic for an Alu element insertion in the ACE gene. The Alu(+/+) genotype occurred 4.5 times more frequently in the control population than the dry/atrophic AMD patient population, (p=0.004). The predominance of the Alu(+/+) genotype within the unaffected control group represents a protective insertion with respect to the human ocular disease, dry/atrophic AMD. This is the first demonstration of an Alu element insertion exerting protective effects against a known human disease.     

Identification of a short interspersed repetitive element insertion polymorphism in the porcine MX1 promoter associated with resistance to porcine reproductive and respiratory syndrome virus infection

The myxovirus resistance (Mx) proteins belong to the dynamin superfamily and are important for innate host defence against RNA viruses. In this study, we demonstrate that positive elements are present in the two promoter regions of ?2713 to ?2565 and ?688 to ?431 in the porcine MX1 gene. Sequencing and alignment of the amplified porcine MX1 gene promoter region identified a short interspersed repetitive element (SINE) insertion of 275 bp at site ?547. At this site, allele B (an insertion of 275 bp) is dominant in Chinese indigenous pig breeds but has a workable minor allele frequency in western lean‐type pig breeds. Luciferase activity was compared between promoters with and without the insertion of the 275‐bp fragment in transiently transfected MARC‐145 cells. The insertion of the 275‐bp fragment increased the luciferase activity significantly (P < 0.05) both prior to and post‐porcine reproductive and respiratory syndrome (PRRS) virus inoculation. These results suggest that the SINE insertion polymorphism at site ?547 of the MX1 gene promoter region is a potential DNA marker for PRRS resistance in pigs.     

Isolation and cloning by a polymerase chain reaction of a genomic DNA fragment of the human slow skeletal troponin (TNNT1) gene

The genomic 3′ structure of the gene coding for the human slow skeletal troponin T (TNNT1) gene, is reported. An intron of 912 nucleotides containing an Alu-element has been identified and characterized. The complexity of the sequenced region suggests an alternative exon use. The present results may be valuable for further studies on the gene structure of TNNT1 and the related troponin gene family.     

Transfer RNA-like structure of the human Alu family: Implications of its generation mechanism and possible functions

Summary Structural resemblance of the human Alu family with a subset of vertebrate tRNAs was detected. Of four tRNAs, tRNA^Lys, tRNA^Ile, tRNA^Thr, and tRNA^Tyr, which comprise a structurally related family, tRNA^Lys is the most similar to the human Alu family. Of the 76 nucleotides in lysine tRNA (including the CCA tail), 47 are similar to the human Alu family (60% identity). The secondary structure of the human Alu family corresponding to the D-stem and anticodon stem regions of the tRNA appears to be very stable. The 7SL RNA, which is a progenitor of the human Alu family, is less similar to lysine tRNA (55% identity), and the secondary structure of the 7SL RNA folded like a tRNA is less stable than that of the human Alu family folded likewise. Insertion of the tetranucleotide GAGA, which is an important region of the second promoter for RNA polymerase III in the Alu sequence, occurred during the deletion and ligation process to generate the Alu sequence from the parental 7SL RNA. These results suggest that the human Alu family was generated from the 7SL RNA by deletion, insertion, and mutations, which thus modified the ancestral 7SL sequence so that it could form a structure more closely resembling lysine tRNA. The similarities of several short interspersed sequences to the lysine tRNA were also examined. TheGalago type 2 family, which was reported to be derived from a methionine initiator tRNA, was also found to be similar to the lysine tRNA. Thus lysine tRNA-like structures are widespread in genomes in the animal kingdom. The implications of these findings in relation to the mechanism of generation of the human Alu family and its possible functions are discussed.     

Porcine Type III RNA Polymerase III Promoters for Short Hairpin RNA Expression

Based on the highly conserved sequences of small nuclear RNA and small cytoplasmic RNA between vertebrate species, three porcine type III RNA polymerase III promoters, pY1, pY3 and pU6, were identified by using genomic DNA walking. To test the functional relationship of these sequences, the human H1 promoter of pSUPER-EGFP-l-neo vector was substituted with these three promoters to create the ppPol III-MCS vectors. The strength of each promoter was measured by its ability to derive expression of shRNA to repress expression of luciferase via RNA interference in the pig kidney epithelial cell line LLC-PK1. We determine that the ranking of promoter strength in descending order is pU6 > pY1 > pY3.