Nuclear copies of mitochondrial genes: another problem for ancient DNA

The application of ancient DNA techniques is subject to many problems caused primarily by low quality and by low quantity of DNA. For these reasons most studies employing ancient DNA rely on the characterization of mitochondrial DNA, which is present in many more copies per cell than nuclear DNA and hence more copies are likely to survive. We used universal and taxon specific mitochondrial primers to amplify DNA from museum specimens, and found many instances where the amplification of nuclear copies of the mitochondrial gene (numts) instead of the targeted mitochondrial fragment had occurred. Furthermore, the likelihood of amplifying numts increased dramatically when universal primers were utilized. Here we suggest that ancient DNA practitioners must consider the possibility that numts can be amplified at higher rates than previously thought. This is another complication for ancient DNA studies, but it also suggests that more extensive inclusion of nuclear markers in ancient DNA studies should be feasible.     

High-mobility group protein 2 may be involved in the locus control region regulation of the beta-globin gene cluster.

Expression regulation of the beta-globin gene cluster is a result of synergistic interactions between cis-elements and trans-acting factors. Previous studies usually concentrated on the core sequence of each hypersensitive site in the locus control region of the beta-globin gene cluster. But more and more evidence illustrates that the flanking regions are indispensable also. Using electrophoretic mobility shift assay and solid-phase DNase I footprinting methods, we identified a small nuclear protein from K562 cells that binds specifically to the first AT-rich region flanking the hypersensitive site 2 core sequence of the human beta-globin gene locus control region. N-terminal sequencing of the enriched protein proved that it is a member of the high-mobility group protein 2 family. This indicates that the AT-rich region in human hypersensitive site 2 may take part in the regulation of the beta-globin gene cluster by facilitating DNA bending, which is a prerequisite for the looping mechanism in this region.     

Beta-globin gene haplotypes in Polynesians are predominantly southern Chinese in type

beta-Globin gene haplotypes obtained in Polynesian Samoans were similar to those described in Southern Chinese. An atypical HindIII restriction fragment length polymorphism detected with pRK29, a 3' beta-globin gene probe, was present at a gene frequency of 7% in Samoans. Haplotype patterns suggest that this polymorphism may have arisen by 1 or 2 mutational events. DNA haplotypes derived from the beta-globin gene cluster confirm nuclear and mitochondrial DNA data that Polynesian precursor populations were East Asian in origin.     

Extensive human DNA contamination in extracts from ancient dog bones and teeth

Ancient DNA (aDNA) sequences, especially those of human origin, are notoriously difficult to analyze due to molecular damage and exogenous DNA contamination. Relatively few systematic studies have focused on this problem. Here we investigate the extent and origin of human DNA contamination in the most frequently used sources for aDNA studies, that is, bones and teeth from museum collections. To distinguish contaminant DNA from authentic DNA we extracted DNA from dog (Canis familiaris) specimens. We monitored the presence of a 148-bp human-specific and a 152-bp dog-specific mitochondrial DNA (mtDNA) fragment in DNA extracts as well as in negative controls. The total number of human and dog template molecules were quantified using real-time polymerase chain reaction (PCR), and the sequences were characterized by amplicon cloning and sequencing. Although standard precautions to avoid contamination were taken, we found that all samples from the 29 dog specimens contained human DNA, often at levels exceeding the amount of authentic ancient dog DNA. The level of contaminating human DNA was also significantly higher in the dog extracts than in the negative controls, and an experimental setup indicated that this was not caused by the carrier effect. This suggests that the contaminating human DNA mainly originated from the dog bones rather than from laboratory procedures. When cloned, fragments within a contaminated PCR product generally displayed several different sequences, although one haplotype was often found in majority. This leads us to believe that recognized criteria for authenticating aDNA cannot separate contamination from ancient human DNA the way they are presently used.     

Optimal Ancient DNA Yields from the Inner Ear Part of the Human Petrous Bone

The invention and development of next or second generation sequencing methods has resulted in a dramatic transformation of ancient DNA research and allowed shotgun sequencing of entire genomes from fossil specimens. However, although there are exceptions, most fossil specimens contain only low (~ 1% or less) percentages of endogenous DNA. The only skeletal element for which a systematically higher endogenous DNA content compared to other skeletal elements has been shown is the petrous part of the temporal bone. In this study we investigate whether (a) different parts of the petrous bone of archaeological human specimens give different percentages of endogenous DNA yields, (b) there are significant differences in average DNA read lengths, damage patterns and total DNA concentration, and (c) it is possible to obtain endogenous ancient DNA from petrous bones from hot environments. We carried out intra-petrous comparisons for ten petrous bones from specimens from Holocene archaeological contexts across Eurasia dated between 10,000-1,800 calibrated years before present (cal. BP). We obtained shotgun DNA sequences from three distinct areas within the petrous: a spongy part of trabecular bone (part A), the dense part of cortical bone encircling the osseous inner ear, or otic capsule (part B), and the dense part within the otic capsule (part C). Our results confirm that dense bone parts of the petrous bone can provide high endogenous aDNA yields and indicate that endogenous DNA fractions for part C can exceed those obtained for part B by up to 65-fold and those from part A by up to 177-fold, while total endogenous DNA concentrations are up to 126-fold and 109-fold higher for these comparisons. Our results also show that while endogenous yields from part C were lower than 1% for samples from hot (both arid and humid) parts, the DNA damage patterns indicate that at least some of the reads originate from ancient DNA molecules, potentially enabling ancient DNA analyses of samples from hot regions that are otherwise not amenable to ancient DNA analyses.     

Detection of Cytosine Methylation in Ancient DNA from Five Native American Populations Using Bisulfite Sequencing

While cytosine methylation has been widely studied in extant populations, relatively few studies have analyzed methylation in ancient DNA. Most existing studies of epigenetic marks in ancient DNA have inferred patterns of methylation in highly degraded samples using post-mortem damage to cytosines as a proxy for cytosine methylation levels. However, this approach limits the inference of methylation compared with direct bisulfite sequencing, the current gold standard for analyzing cytosine methylation at single nucleotide resolution. In this study, we used direct bisulfite sequencing to assess cytosine methylation in ancient DNA from the skeletal remains of 30 Native Americans ranging in age from approximately 230 to 4500 years before present. Unmethylated cytosines were converted to uracils by treatment with sodium bisulfite, bisulfite products of a CpG-rich retrotransposon were pyrosequenced, and C-to-T ratios were quantified for a single CpG position. We found that cytosine methylation is readily recoverable from most samples, given adequate preservation of endogenous nuclear DNA. In addition, our results indicate that the precision of cytosine methylation estimates is inversely correlated with aDNA preservation, such that samples of low DNA concentration show higher variability in measures of percent methylation than samples of high DNA concentration. In particular, samples in this study with a DNA concentration above 0.015 ng/μL generated the most consistent measures of cytosine methylation. This study presents evidence of cytosine methylation in a large collection of ancient human remains, and indicates that it is possible to analyze epigenetic patterns in ancient populations using direct bisulfite sequencing approaches.     

The presence of ancient human T-cell lymphotropic virus type I provirus DNA in an Andean mummy

The worldwide geographic and ethnic clustering of patients with diseases related to human T-cell lymphotropic virus type I (HTLV-I) may be explained by the natural history of HTLV-I infection. The genetic characteristics of indigenous people in the Andes are similar to those of the Japanese, and HTLV-I is generally detected in both groups. To clarify the common origin of HTLV-I in Asia and the Andes, we analyzed HTLV-I provirus DNA from Andean mummies about 1,500 years old. Two of 104 mummy bone marrow specimens yielded a band of human beta-globin gene DNA 110 base pairs in length, and one of these two produced bands of HTLV-I-pX (open reading frame encoding p40x, p27x) and HTLV-I-LTR (long terminal repeat) gene DNA 159 base pairs and 157 base pairs in length, respectively. The nucleotide sequences of ancient HTLV-I-pX and HTLV-I-LTR clones isolated from mummy bone marrow were similar to those in contemporary Andeans and Japanese, although there was microheterogeneity in the sequences of some mummy DNA clones. This result provides evidence that HTLV-I was carried with ancient Mongoloids to the Andes before the Colonial era. Analysis of ancient HTLV-I sequences could be a useful tool for studying the history of human retroviral infection as well as human prehistoric migration.     

Roles of fetal G gamma-globin promoter elements and the adult beta-globin 3'' enhancer in the stage-specific expression of globin genes.

The human fetal G gamma-globin and adult beta-globin genes are expressed in a tissue- and developmental stage-specific pattern in transgenic mice: the G gamma gene in embryonic cells and the beta gene in fetal and adult erythroid cells. Several of the cis-acting DNA sequences thought to be responsible for these patterns of expression are located 5' to the G gamma-globin gene and 3' to the beta-globin gene. To further define the locations and functional roles of these elements, we examined the effects of 5' truncations on the expression of the G gamma-globin gene, as well as the ability of G gamma-globin upstream sequences to alter the developmental regulation of a beta-globin gene, as well as the ability of G gamma-globin upstream sequences to alter the developmental regulation of a beta-globin gene. We found that sequences between -201 and -136 are essential for expression of the G gamma-globin gene, whereas those upstream of -201 have little effect on the level or tissue or stage specificity of G gamma-globin expression. The G gamma-globin upstream sequences from -201 to -136 were, furthermore, capable of activating a linked beta-globin gene in embryonic blood cells; however, a G gamma-globin fragment from -383 to -206 was similarly active in this assay, and the complete fragment from -383 to -136 was considerably more active than either of the smaller fragments, suggesting the presence of multiple cis-acting elements for embryonic blood cells. Our data also suggested the possibility of a negative regulatory element between -201 and -136. These results are discussed in relation to several DNA elements in the G gamma-globin upstream region, which have been shown to bind nuclear factors in erythroid cells. Finally, we observed that removal of the beta-globin 3'-flanking sequences, including the 3' enhancer, from the G gamma-globin upstream-beta-globin hybrid gene resulted in a 25-fold reduction in expression in embryonic blood cells. This suggests that the beta-globin 3' enhancer is potentially active at the embryonic stage and thus cannot be solely responsible for the fetal or adult specificity of the beta-globin gene.     

Multiple sequence-specific DNA binding activities are eluted from chicken nuclei at low ionic strengths.

DNA sequence-specific binding proteins eluted from chicken erythrocyte and thymus nuclei, and fractionated as described by Emerson and Felsenfeld (19), have been investigated by filter binding and footprint analyses. The erythrocyte nuclear protein fraction specifically binds to at least two sites within the 5' flanking chromatin hypersensitive site of the chicken beta A-globin gene, and to a site 5' to the human beta-globin gene. The major chicken beta A globin gene binding site [G)18CGGGTGG) and the human beta-globin gene binding site [TA)6(T)8C(T)4) occur at or near sequences which are hypersensitive to S1 nuclease cleavage in supercoiled plasmids. Downstream, the second chicken beta A-globin gene binding site includes the beta-globin gene CACCC consensus sequence. Filter binding studies also show other sequence specific binding activities to human N-ras and human (but not chicken) c-myc gene sequences.     

The nucleotide sequence of the human beta-globin gene

We report the complete nucleotide sequence of the human beta-globin gene. The purpose of this study is to obtain information necessary to study the evolutionary relationships between members of the human beta-like globin gene family and to provide the basis for comparing normal beta-globin genes with those obtained from the DNA of individuals with genetic defects in hemoglobin expression.     

Detection of a Tumor Suppressor Gene Variant Predisposing to Colorectal Cancer in an 18th Century Hungarian Mummy

Mutations of the Adenomatous polyposis coli (APC) gene are common and strongly associated with the development of colorectal adenomas and carcinomas. While extensively studied in modern populations, reports on visceral tumors in ancient populations are scarce. To the best of our knowledge, genetic characterization of mutations associated with colorectal cancer in ancient specimens has not yet been described. In this study we have sequenced hotspots for mutations in the APC gene isolated from 18^th century naturally preserved human Hungarian mummies. While wild type APC sequences were found in two mummies, we discovered the E1317Q missense mutation, known to be a colorectal cancer predisposing mutation, in a large intestine tissue of an 18^th century mummy. Our data suggests that this genetic predisposition to cancer already existed in the pre-industrialization era. This study calls for similar investigations of ancient specimens from different periods and geographical locations to be conducted and shared for the purpose of obtaining a larger scale analysis that will shed light on past cancer epidemiology and on cancer evolution.     

Upstream G gamma-globin and downstream beta-globin sequences required for stage-specific expression in transgenic mice.

The human G gamma-globin and beta-globin genes are expressed in erythroid cells at different stages of human development, and previous studies have shown that the two cloned genes are also expressed in a differential stage-specific manner in transgenic mice. The G gamma-globin gene is expressed only in murine embryonic erythroid cells, while the beta-globin gene is active only at the fetal and adult stages. In this study, we analyzed transgenic mice carrying a series of hybrid genes in which different upstream, intragenic, or downstream sequences were contributed by the beta-globin or G gamma-globin gene. We found that hybrid 5'G gamma/3'beta globin genes containing G gamma-globin sequences upstream from the initiation codon were expressed in embryonic erythroid cells at levels similar to those of an intact G gamma-globin transgene. In contrast, beta-globin upstream sequences were insufficient for expression of 5'beta/3'G gamma hybrid globin genes or a beta-globin-metallothionein fusion gene in adult erythroid cells. However, beta-globin downstream sequences, including 212 base pairs of exon III and 1,900 base pairs of 3'-flanking DNA, were able to activate a 5'G gamma/3'beta hybrid globin gene in fetal and adult erythroid cells. These experiments suggest that positive regulatory elements upstream from the G gamma-globin and downstream from the beta-globin gene are involved in the differential expression of the two genes during development.     

Nucleotide sequence analysis of the delta beta-globin gene region in humans

The continuous DNA sequence of a 16.5-kilobase pair region encompassing the linked delta beta-globin gene cluster in humans is presented with a detailed restriction endonuclease map. There are 38 differences (0.5%) in comparison with published sequence data, corrected for errors in sequencing, resulting in polymorphic rates of 0.2% in exons and 0.76% in 5'-gene flanking regions. Fifteen changes result in the generation or elimination of restriction sites which may be useful in linkage disequilibrium studies. Two pairs of inverted Alu repeats, a pyrimidine-rich region 5' to delta, and (TG)n, (Pu/Py)n, and (ATTTT)n tracts 5' to beta are described. Dinucleotide frequencies and deviation from expected values approximated those found in total human genomic DNA. Regions of less than 50% A + T content were found associated with Alu sequences, a 150-base pair region immediately 5' to the beta gene, exon regions from both genes, and an area 3' to the beta gene. These regions also contained significantly lower than expected CpG levels compared to other regions, suggesting a possible relationship between DNA organizational patterns and functionally important regions. In addition, strand asymmetries in base composition in this region differ from those associated with the fetal globin genes.     

In vitro characterization of tissue-specific nuclear proteins preferentially bound to the mouse beta-globin gene during MEL cell terminal differentiation

Using DNA restriction fragments of the mouse beta-globin gene and other promoter-containing DNA fragments (LTR-MMTV and pBR322) as controls, we have characterized by protein blotting, in extracts of mouse erythroleukemia (MEL) cells, specific nuclear DNA binding proteins with a preferential affinity for the beta-globin DNA. Some proteins (110 and 75 kDa) appear in differentiated MEL cells while others (100, 95, and 35 kDa) are present in immature MEL and normal erythroblast cells and bind selectively to the far-upstream region of the gene. These proteins could modulate either positively or negatively the expression of the beta-globin gene and maybe, of other genes, during the terminal differentiation of erythroid cells.     

A review on the origin and spread of deleterious mutants of the β-globin gene in Indian populations

Deleterious mutations of the human beta-globin gene are responsible for beta-thalassaemia and other haemoglobinopathies, which are the most common genetic diseases in Indian populations. A highly heterogeneous distribution of those mutations is observed in India and certain mutations are restricted to some extent to particular groups only. The reasons behind the geographical clustering and origin of the mutations in India is a highly debated issue and the evidence is conflicting. Our present article aims at tracing the origin of the deleterious beta-globin mutation and evaluates the role of different evolutionary forces responsible for the spread and present distribution of those mutations in Indian populations, using data from molecular biology and statistical methods. Mutations are generated essentially randomly, but "hot-spot" sites for mutation are reported for the beta-globin gene cluster, indicating sequence dependency of mutation. A single origin of a deleterious beta-globin mutation, followed by recombination (in a hot spot region) and/or interallelic gene conversion (within beta-globin gene) through time is the most plausible hypothesis to explain the association of those mutations with multiple haplotype backgrounds and frameworks. It is suggested that India is the place of origin of HbE and HbD mutations and that they dispersed to other parts of the would by migration. HbS mutants present in Indian populations are not of Middle East origin but rather a fresh mutation is the probable explanation for the prevalence among tribal groups. beta-thalassaemia represents a heterogeneous group of mutant alleles in India. Five common and twelve rare mutations have been reported in variable frequencies among different Indian populations. Gene flow of those mutant alleles from different populations of the world by political, military and commercial interactions possibly accounts for the heterogenous nature of beta-thalassaemia among Indians. A multiple allelic polymorphic system of the beta-globin gene exists in different populations. Dynamic interaction of the mutant alleles in the presence of different selective forces including falciparum malaria and biosocial patterns of Indian populations is discussed in order to explain the variable distribution and maintenance of those mutant alleles.