Molecular, forensic and haplotypic inconsistencies regarding the identity of the Ekaterinburg remains.

BACKGROUND: A set of human remains unearthed near Ekaterinburg, Russia has been attributed to the Romanov Imperial Family of Russia and their physician and servants. That conclusion was officially accepted by the Russian government following publication of DNA tests that were widely publicized. The published study included no discussion of major forensic discrepancies and the information regarding the burial site and remains included irregularities. Furthermore, its conclusion of Romanov identity was based on molecular behaviour that indicates contamination rather than endogenous DNA. The published claim to have amplified by PCR a 1223 bp region of degraded DNA in a single segment for nine individuals and then to have obtained sequence of PCR products derived from that segment without cloning indicates that the Ekaterinburg samples were contaminated with non-degraded, high molecular weight, 'fresh' DNA. AIM: Noting major violations of standard forensic practices, factual inconsistencies, and molecular behaviours that invalidate the claimed identity, we attempted to replicate the findings of the original DNA study. SUBJECT: We analysed mtDNA extracted from a sample of the relic of Grand Duchess Elisabeth, sister of Empress Alexandra. RESULTS: Among clones of multiple PCR targets and products, we observed no complete mtDNA haplotype matching that reported for Alexandra. The consensus haplotype of Elisabeth differs from that reported for Alexandra at four sites. CONCLUSION: Considering molecular and forensic inconsistencies, the identity of the Ekaterinburg remains has not been established. Our mtDNA haplotype results for Elisabeth provide yet another line of conflicting evidence regarding the identity of the Ekaterinburg remains.

Evolutionary history of B1 retroposons in the genus Mus.

Short interspersed DNA elements (SINEs) amplify by retroposition either by (i) successive waves of amplification from one or a few evolving master genes or by (ii) the generation of new master genes that coexist with their progenitors. Individual, highly conserved, elements of the B1 SINE family were identified from the GenBank nucleotide database using various B1 subfamily consensus query sequences to determine their integration times into the mouse genome. A comparison of orthologous loci in various species of the genus Mus demonstrated that four subfamilies of B1 elements have been amplifying within the last 1-3 million years. Therefore, B1 sequences are generated by coexisting source genes. Additionally, three B1 subfamilies have been concurrently propagated during subspecies divergence and strain formation in Mus, indicating very recent activity of this retroposon family. The patterns of intra- and interspecies variations of orthologous loci demonstrate the usefulness of B1 integrations as a phylogenetic tool. A single inconsistency in the phylogenetic trends was depicted by the presence of a B1 insert in an orthologous locus exclusively in M. musculus and M. pahari. However, DNA sequence analysis revealed that these were independent integrations at the same genomic site. One highly conserved B1 element that integrated at least 4-6 million years ago suggests the possibility of occasional function for B1 integrations.

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.

Recent B2 element insertions in the mouse genome.

B2 elements are a family of short interspersed repeats that have amplified within rodent genomes. Recent mobility of only two individual B2 elements has been reported to date. We identified an additional recent B2 insertion occurring within intron 4 of the murine beta-glucuronidase gene (Gus-s) of the BalbC strain of mouse by analyzing orthologous loci of a nonrandomly selected B2 element. The basis of selection for the B2 element was its high level of sequence identity to the B2 consensus. The selected B2 element was amplified by the polymerase chain reaction (PCR) using primers to the unique flanking sequences from genomic DNA of several species and laboratory strains of mice. Our results demonstrated the presence of the selected B2 element only in the genome of Mus musculus BalbC strain. Cloning and sequencing of a representative sample of the products obtained confirmed the absence of the B2 element within this intron in addition to other variations in the sequence. The detection of the B2 element only in the BalbC strain suggests that the element recently inserted within this mouse population when the initial laboratory colony was formed. Sequence comparison of the two previously identified recent B2 inserts also shows a low divergence in relation to the B2 type II consensus. The data presented confirms that recently inserted B2 elements closely match their consensus sequence, potentially allowing for their identification.

Evolution of B2 repeats: the muroid explosion.

B2 repeats are a group of short interspersed elements (SINEs) specific for rodent genomes. Copy numbers were determined for different rodent genera. All the Muroid (rat, mouse, deer mouse, hamster, gerbil) rodent genomes analyzed exhibited 80,000-100,000 copies per haploid genome, whereas the squirrel genome contains only 2,500 copies, and fewer than 100 (if any) copies were observed for the Hystricognath rodents (guinea pig and nutria). These findings demonstrate that there was an 'explosion' of amplification of B2 elements within muroid rodents. The similar copy number of B2 elements within the different muroid species could be explained by formation of a high proportion of the B2 elements prior to the divergence of the different muroid species. However, the 3'-end of the B2 sequence is unique between murid and cricetid rodents suggesting that the majority of elements amplified after the divergence of these species. Also consistent with recent amplification of these elements in parallel within the muroid genomes is the finding that within mouse and rat there are distinct subfamilies of B2 repeats. The pattern of consistent parallel amplification of B2 elements in muroid species contrasts with the sporadic nature of ID repeat amplification in the same genomes. The consensus of the young mouse subfamily of elements corresponds to the B2 RNA that is preferentially transcribed in embryonic, tumor, and normal liver cells. The subfamily is young based on both its low divergence from the subfamily consensus sequence and the finding that the most recent B2 element insertions in the mouse genome are members of this subfamily.

Characterization and population diversity of interspersed repeat sequence variants (IRS-morphs).

Inter-Alu PCR is increasingly useful in human genome mapping studies. One use is the generation of alumorphs, polymorphisms resulting from the presence or absence of inter-Alu PCR products. In this study, we have increased the proportion of the genome that can be analyzed by this technique with the use of long interspersed elements (LINEs). The set of polymorphisms detected by both Alu and LINE primers are referred to as interspersed repetitive sequence variants or IRS-morphs. Since a presence-absence variant may have been the result of a recent Alu or LINE insertion, we analyzed 7 isolated IRS-morphs that were generated, in part, with a primer derived from either a consensus LINE or a young Alu subfamily specific sequence, and observed by Southern blot analysis that these variants resulted from other types of genomic alterations. The use of these primers, however, reduces background from the numerous LINEs and Alu elements in the genome, providing sharp DNA fingerprint profiles. We have demonstrated the potential usefulness of these IRS-morph profiles in human population studies. We compared 12 IRS-morphs from a single amplification reaction from five distinct population groups (Caucasian (northern European descent), Hispanic (Mexican-American), Hindu-Indian, Papua New Guinean, and Greenland Eskimo) and observed that most have variable allelic frequencies among populations. The utilization of additional IRS-morph profiles will perpetuate this technique as a tool for DNA fingerprinting and for the analysis of human populations. Key words : Alu elements, DNA fingerprint, human populations, LINEs, SINEs.

Genetic variation of recent Alu insertions in human populations.

The Alu family of interspersed repeats is comprised of over 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 insertions 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 population groups.

Sporadic amplification of ID elements in rodents.

ID sequences are members of a short interspersed element (SINE) repetitive DNA family within the rodent genome. The copy number of individual ID elements varies by up to three orders of magnitude between species. This amplification has been highly sporadic in the order Rodentia and does not follow any phylogenetic trend. Using library screening and dot-blot analysis, we estimate there are 25,000 copies of ID elements in the deer mouse, 1,500 copies in the gerbil (both cricetid rodents), and 60,000 copies of either ID or ID-like elements in a sciurid rodent (squirrel). By dot-blot analysis, we estimate there are 150,000, 4,000, 1,000, and 200 copies of ID elements in the rat, mouse, hamster, and guinea pig, respectively (which is consistent with previous reports) and 200 copies in the hystricognath rodent, nutria. Therefore, a rapid amplification took place not only after the divergence of rat and mouse but also following the deer mouse (Peromyscus) and hamster split, with no evidence of increased amplifications in hystricognath rodents. No notable variations of sequences from the BC1 genes of several myomorphic rodents were observed that would possibly explain the varied levels of ID amplification. We did observe subgenera and species-group-specific variation in the ID core sequence of the BC1 gene within the genus Peromyscus. Sequence analysis of cloned ID elements in Peromyscus show most ID elements in this genus arose prior to Peromyscus subgenus divergence. Correspondence of the consensus sequence of individual ID elements in gerbil and deer mouse further confirms BC1 as a master gene in ID amplification. Several possible mechanisms responsible for the quantitative variations are explored.

Transcription and processing of the rodent ID repeat family in germline and somatic cells.

ID elements comprise a rodent SINE (short interspersed DNA repetitive element) family that has amplified by retroposition of a few master genes. In order to understand the important factors of SINE amplification, we investigated the transcription of rat ID elements. Three different size classes of ID transcripts, BC1, BC2 and T3, have been detected in various rat tissues, including brain and testes. We have analysed the nucleotide sequences of testes- and brain-derived ID transcripts isolated by size-fractionation, C-tailing and RACE. Nucleotide sequence variation of testes ID transcripts demonstrated derivation from different loci. However, the transcripts represent a preferred set of ID elements that closely match the subfamily consensus sequences. The small ID transcripts, T3, are not comprised of primary transcripts, but are instead processed polyA-transcripts generated from many different loci. These truncated transcripts would be expected to be retroposition-incompetent forms. Therefore, the amplification of ID elements is likely to be regulated at multiple steps of retroposition, which include transcription and processing. Although brain ID transcripts showed a similar pattern, with the addition of very high levels of transcription from the BC1 locus, we also found evidence that a single locus dominated the production of brain BC2 RNA species. BC1 RNA is highly stable in both germ line and brain cells, based on the low level of detection of the processing product, T3. This stability of BC1 RNA might have been a contributing factor in its role as a master gene for ID amplification.

A new restriction-site polymorphism in exon 18 of the low density lipoprotein receptor (LDLR) gene.

A new restriction fragment length polymorphism (RFLP) in exon 18 of the low density lipoprotein receptor (LDLR) gene is described. It should be a useful marker in linkage to familial hypercholesterolemia.

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.

African origin of human-specific polymorphic Alu insertions.

Alu elements are a family of interspersed repeats that have mobilized throughout primate genomes by retroposition from a few "master" genes. Among the 500,000 Alu elements in the human genome are members of the human-specific subfamily that are not fixed in the human species; that is, not all chromosomes carry an Alu element at a particular locus. Four such polymorphic human-specific Alu insertions were analyzed by a rapid, PCR-based assay that uses primers that flank the insertion point to determine genotypes based on the presence or absence of the Alu element. These four polymorphic Alu insertions were shown to be absent from the genomes of a number of nonhuman primates, consistent with their arising as human genetic polymorphisms sometime after the human/African ape divergence. Analysis of 664 unrelated individuals from 16 population groups from around the world revealed substantial levels of variation within population groups and significant genetic differentiation among groups. No significant associations were found among the four loci, consistent with their location on different chromosomes. A maximum-likelihood tree of population relationships showed four major groupings consisting of Africa, Europe, Asia/Americas, and Australia/New Guinea, which is concordant with similar trees based on other loci. A particularly useful feature of the polymorphic Alu insertions is that the ancestral state is known to be the absence of the Alu element, and the presence of the Alu element at a particular chromosomal site reflects a single, unique event in human evolution. A hypothetical ancestral group can then be included in the tree analysis, with the frequency of each insertion set to zero. The ancestral group connected to the maximum-likelihood tree within the African branch, which suggests an African origin of these polymorphic Alu insertions. These data are concordant with other diverse data sets, which lends further support to the recent African origin hypothesis for modern humans. Polymorphic Alu insertions represent a source of genetic variation for studying human population structure and evolution.

Comparison of chromosomal distribution of a retroposon (LINE) and a retrovirus-like element mys in Peromyscus maniculatus and P. leucopus.

Chromosomal distribution for two interspersed elements (LINEs and mys) that are thought to have established their chromosomal position primarily by transposition was compared between two species of deer mice (Peromyscus leucopus and P. maniculatus). Both LINEs and mys generally produced an autosomal banding pattern reflective of G-bands and both hybridized preferentially to the sex chromosomes. The pattern on the long arm of the X was unique for each, with mys reflecting the G-bands (four bands with the telomeric most prominent) and LINE producing five equally spaced bands of equal intensity. LINE also preferentially hybridized to the short arm of the longest autosomal pair. Some aspects of these patterns are explained adequately with proposed mechanisms that would produce a non-random pattern of chromosomal distribution (i.e. both reflect autosomal G-bands and both preferentially insert into AT-rich regions characteristic of G-bands). However, other aspects such as the differences observed on the long arm of the X do not appear to fit any predictions of proposed mechanisms.

Identification of a human specific Alu insertion in the factor XIIIB gene.

The factor XIIIB gene was examined to determine the nature of a previously described 300 bp restriction fragment length polymorphism (RFLP) seen in the human population. Polymerase chain reaction analysis of different regions within the factor XIIIB gene was carried out to define a high resolution map of the region encompassing the polymorphism, followed by DNA sequence analysis. An Alu insertion was found to be the source of this variation. This Alu repeat is a member of the human specific-1 (HS-1) subfamily, although one of the five diagnostic nucleotides is a cattarhine specific (CS) subfamily mutation, suggesting that it may represent an intermediate form in the evolution between these two subfamilies. Subsequently, we developed a PCR-based assay to detect the polymorphism, rendering it a more useful marker for genetic linkage studies and genome mapping. This insertion is also a valuable polymorphism for human population studies, as demonstrated by the large variations in allele frequencies seen in three population groups.

Examination of DNA methylation of chromosomal hot spots associated with breast cancer.

Widespread hypomethylation of DNA and regional hypermethylation, including tumor suppressor regions, have been demonstrated in several human cancers. Since a highly heterogeneous array of genetic anomalies have been associated with breast cancer, we examined several chromosomal hot spots for abnormal methylation patterns. Low-levels of increased methylation of HRAS (11p15) were observed between normal and tumor breast tissue samples from 8 patients. No noticeable variation in methylation was observed with DNA probes from chromosomes 11p15, 1p36, 17q22, 17p13.3 and 3p21 for the 7 ductal breast carcinoma patients, though some variability was observed for a patient with atypical medullary carcinoma. Additionally, the methylation pattern of the estrogen-receptor gene (6q24-27), whose protein product is increased in numerous breast cancers, also did not change. Therefore, as opposed to other cancer types, widespread hypomethylation and regional hypermethylation do not appear to be involved in the early stages of breast cancer and does not account for the molecular heterogeneity of the disease. Proposed alternative mechanisms for the diversity of genetic alterations associated with breast cancer are discussed.

Unusual patterns of susceptibility to degradation of DNA isolated from tissues in Peromyscus californicus.

Isolation of intact, high molecular weight genomic DNA from the livers of 2 subspecies of Peromyscus californicus without excessive degradation was typically unattainable, whereas highly intact DNA from livers of other Peromyscus (field mice) species is invariably obtained using the same isolation methods. Additionally, highly intact DNA was obtained from splenic tissues of adult P. californicus and hepatic tissue of juvenile animals, indicating that the phenomenon is tissue-specific and age-related.

Growth regulation in Peromyscus species hybrids: a test for mitochondrial-nuclear genomic interaction.

Interspecific hybridization of Peromyscus maniculatus (deer mouse) and P. polionotus (oldfield mouse) is accompanied by pronounced size differences between reciprocal F1 animals beginning in the fetus and continuing throughout life. Since the mitochondrial genome is inherited through the maternal line in Peromyscus, we tested the hypothesis that increased disparity between the species sources of mitochondrial and nuclear genomes within animals would exaggerate the reciprocal size effects through misregulation of growth, whereas species-compatible genomes were postulated to diminish the effect. Four series of backcrosses were established from females of the two reciprocal F1, while insuring continuity of the maternal mitochondrial composition at each generation. Size and weight measurements were made on neonatal, ten-day and six-month old animals through four or more backcross generations. Contrary to the hypothesis, deer mice with P. polionotus mitochondrial DNA, but 98% or more P. maniculatus nuclear composition, and animals with P. maniculatus mitochondria and principally P. polionotus nuclear genome regressed in mean size parameters to those of P. maniculatus and P. polionotus, respectively. Most of the regression was accounted for by the second backcross generation, and second and later backcross progeny did not differ significantly from the respective parental species controls. Maternal inheritance of mitochondrial DNA was confirmed by restriction enzyme analysis at the second and fifth backcross generation. Hybrid maternal effects in this Peromyscus cross are likely attributable to mechanisms other than nuclear-mitochondrial genomic interaction.

The evolution of coexisting highly divergent LINE-1 subfamilies within the rodent genus Peromyscus.

Two distinct members of the LINE-1 (L1) family in Peromyscus were characterized. The two clones, denoted L1Pm55 and L1Pm62, were 1.5 kb and 1.8 kb in length, respectively, and align to the identical region of the L1 sequence of Mus domesticus. Sequence similarity was on the order of 70% between L1Pm55 and L1Pm62, which approximates that between either Peromyscus sequence and Mus L1. L1Pm62 represents a more prevalent subfamily than L1Pm55. L1Pm62 exists in about 500 copies per haploid genome, while L1Pm55 exists in about 100 copies. The existence of major and minor subpopulations of L1 within Peromyscus is in contrast to murine rodents and higher primates, where L1 copy number is on the order of 20,000 to 100,000, and where levels of intraspecific divergence among L1 elements are typically less than 15-20%. Additional Peromyscus clones are similarly divergent from both L1Pm62 and L1Pm55, implying the existence of more than two distinct L1 subfamilies. The highly divergent L1 subfamilies in Peromyscus apparently have been evolving independently for more than 25 million years, preceding the divergence of cricetine and murine rodents. Investigations of the evolution of L1 within Peromyscus by restriction and Southern analysis was performed using species groups represented by the partially interfertile species pairs P. maniculatus-P. polionotus, P. leucopus-P. gossypinus, and P. truei-P. difficilis of the nominate subgenus and P. californicus of the Haplomylomys subgenus. Changes in L1 and species group taxonomic boundaries frequently coincided. The implications for phylogeny are discussed.