Prion infected rhesus monkeys to study differential transcription of Alu DNA elements and editing of Alu transcripts in neuronal cells and blood cells.

BACKGROUND: Rhesus monkeys were used as a non-human primate model to study small non-coding RNA after infection with human sporadic and variant Creutzfeldt-Jakob prions. METHODS: Tissue-specific Alu DNA element transcription and editing of transcripts were assessed in neuronal - and blood cells (Buffy Coat). RESULTS: Tissue/cell-specific transcription and editing patterns were obtained. Active Alu DNA elements belonged to several Alu DNA families, they could be located on several chromosomes, and their genomic sites were identified. Deamination by adenosine deaminase acting on RNA and apolipoprotein B editing complex was found. CONCLUSIONS: Different Alu transcription and editing programmes exist and may depend on the infection status.

Methylation and expression analyses of the 7q autism susceptibility locus genes MEST , COPG2, and TSGA14 in human and anthropoid primate cortices.

The autism susceptibility locus on human chromosome 7q32 contains the maternally imprinted MEST and the non-imprinted COPG2 and TSGA14 genes. Autism is a disorder of the 'social brain' that has been proposed to be due to an overbalance of paternally expressed genes. To study regulation of the 7q32 locus during anthropoid primate evolution, we analyzed the methylation and expression patterns of MEST, COPG2, and TSGA14 in human, chimpanzee, Old World monkey (baboon and rhesus macaque), and New World monkey (marmoset) cortices. In all human and anthropoid primate cortices, the MEST promoter was hemimethylated, as expected for a differentially methylated imprinting control region, whereas the COPG2 and TSGA14 promoters were completely demethylated, typical for transcriptionally active non-imprinted genes. The MEST gene also showed comparable mRNA expression levels in all analyzed species. In contrast, COPG2 expression was downregulated in the human cortex compared to chimpanzee, Old and New World monkeys. TSGA14 either showed no differential regulation in the human brain compared to chimpanzee and marmoset or a slight upregulation compared to baboon. The human-specific downregulation supports a role for COPG2 in the development of a 'social brain'. Promoter methylation patterns appear to be more stable during evolution than gene expression patterns, suggesting that other mechanisms may be more important for inter-primate differences in gene expression.

Hybridization between mouse lemurs in an ecological transition zone in southern Madagascar.

Hybrid zones in ecotones can be useful model systems for the study of evolutionary processes that shape the distribution and discreteness of species. Such studies could be important for an improved understanding of the complex biogeography of Madagascar, which is renowned for its outstanding degree of small-scale endemism. Certain forest remnants in central Madagascar indicate that transitional corridors across the island could have connected microendemics in different forest types in the past. Evolutionary processes in such corridors are difficult to study because most of these corridors have disappeared due to deforestation in central Madagascar. We studied a hybrid zone in one of the few remaining ecotonal corridors between dry and humid forests in Madagascar, which connects two species of mouse lemurs, Microcebus griseorufus in dry spiny forest and Microcebus murinus in humid littoral forest. We sampled 162 mouse lemurs at nine sites across this boundary. Morphometric analyses revealed intermediate morphotypes of many individuals in transitional habitat. Bayesian clustering of microsatellite genotypes and assignment tests yielded evidence for a mixed ancestry of mouse lemurs in the ecotone, where we also observed significant linkage disequilibria and heterozygote deficiency. In contrast to these observations, mitochondrial haplotypes displayed a sharply delimited boundary at the eastern edge of spiny forest, which was noncoincident with the signals from microsatellite data. Among several alternative scenarios, we propose asymmetric nuclear introgression due to male-biased dispersal, divergent environmental selection, and an expansion of dry spiny forest in the course of aridification as a probable explanation of our observations.

Primate genomes.

A particular interest in primate genetics was fueled by the release of the complete human genome sequence drafts reported in 2001 by the IHGSC and Celera Genomics. Postgenomic comparative analyses based on the complete genome sequence of the mouse started focusing on functional, evolutionary and diversity aspects of human DNA. By analyzing molecular character states in the representatives of the major primate groups one is able to reconstruct the processes that shape genomes on the lineage to humans after the mouse-human divergence. Consequently, several primate genome sequences are about to be generated during Whole Genome Shotgun (WGS) sequencing projects and are already available for two representatives of the Old World monkeys and hominoids (rhesus monkey, chimpanzee). Comparative data restricted to functional genome parts of a meaningful primate sample (ENCODE project) are underway. These data will yield a definite phylogenetic framework linking the mouse, primate related eutherians and the major primate groups, which is indispensable for any analysis of character evolution. Concerning the functional site comparative genetic research in primates on molecular phenomena that control the spatiotemporal profile of the cellular RNA and protein composition will contribute to our understanding of genotype-phenotype correlations and the emergence of human specific traits.

Primate phylogeny: molecular evidence from retroposons.

In these postgenomic times where aspects of functional genetics and character evolution form a focal point of human-mouse comparative research, primate phylogenetic research gained a widespread interest in evolutionary biology. Nevertheless, it also remains a controversial subject. Despite the surge in available primate sequences and corresponding phylogenetic interpretations, primate origins as well as several branching events in primate divergence are far from settled. The analysis of SINEs - short interspersed elements - as molecular cladistic markers represents a particularly interesting complement to sequence data. The following summarizes and discusses potential applications of this new approach in molecular phylogeny and outlines main results obtained with SINEs in the context of primate evolutionary research. Another molecular cladistic marker linking the tarsier with the anthropoid primates is also presented. This eliminates any possibility of confounding phylogenetic interpretations through lineage sorting phenomena and makes use of a new point of view in settling the phylogenetic relationships of the primate infraorders.

HERV-K(OLD): ancestor sequences of the human endogenous retrovirus family HERV-K(HML-2).

Sequences homologous to the human endogenous retrovirus (HERV) family HERV-K(HML-2) are present in all Old World primate species. A previous study showed that a central region of the HERV-K(HML-2) gag genes in Hominoidea species displays a 96-bp deletion compared to the gag genes in lower Old World primates. The more ancient HERV-K(HML-2) sequences present in lower Old World primates were apparently not conserved during hominoid evolution, as opposed to the deletion variants. To further clarify the evolutionary origin of the HERV-K(HML-2) family, we screened GenBank with the 96-bp gag-sequence characteristic of lower Old World primates and identified, to date, 10 human sequence entries harboring either full-length or partially deleted proviral structures, probably representing remnants of a more ancient HERV-K(HML-2) variant. The high degree of mutations demonstrates the long-time presence of these HERV-K(OLD) proviruses in the genome. Nevertheless, they still belong to the HML-2 family as deduced from dot matrix and phylogenetic analyses. We estimate, based on the family ages of integrated Alu elements and on long terminal repeat (LTR) divergence data, that the average age of HERV-K(OLD) proviruses is ca. 28 million years, supporting an integration time before the evolutionary split of Hominoidea from lower Old World primates. Analysis of HERV-K(OLD) LTR sequences led to the distinction of two subgroups, both of which cluster with LTRs belonging to an evolutionarily older cluster. Taken together, our data give further insight into the evolutionary history of the HERV-K(HML-2) family during primate evolution.

SINE insertions in cladistic analyses and the phylogenetic affiliations of Tarsius bancanus to other primates.

Transpositions of Alu sequences, representing the most abundant primate short interspersed elements (SINE), were evaluated as molecular cladistic markers to analyze the phylogenetic affiliations among the primate infraorders. Altogether 118 human loci, containing intronic Alu elements, were PCR analyzed for the presence of Alu sequences at orthologous sites in each of two strepsirhine, New World and Old World monkey species, Tarsius bancanus, and a nonprimate outgroup. Fourteen size-polymorphic amplification patterns exhibited longer fragments for the anthropoids (New World and Old World monkeys) and T. bancanus whereas shorter fragments were detected for the strepsirhines and the outgroup. From these, subsequent sequence analyses revealed three Alu transpositions, which can be regarded as shared derived molecular characters linking tarsiers and anthropoid primates. Concerning the other loci, scenarios are represented in which different SINE transpositions occurred independently in the same intron on the lineages leading both to the common ancestor of anthropoids and to T. bancanus, albeit at different nucleotide positions. Our results demonstrate the efficiency and possible pitfalls of SINE transpositions used as molecular cladistic markers in tracing back a divergence point in primate evolution over 40 million years old. The three Alu insertions characterized underpin the monophyly of haplorhine primates (Anthropoidea and Tarsioidea) from a novel perspective.

The complete mitochondrial genome of Tupaia belangeri and the phylogenetic affiliation of scandentia to other eutherian orders.

The complete mitochondrial genome of Tupaia belangeri, a representative of the eutherian order Scandentia, was determined and compared with full-length mitochondrial sequences of other eutherian orders described to date. The complete mitochondrial genome is 16, 754 nt in length, with no obvious deviation from the general organization of the mammalian mitochondrial genome. Thus, features such as start codon usage, incomplete stop codons, and overlapping coding regions, as well as the presence of tandem repeats in the control region, are within the range of mammalian mitochondrial (mt) DNA variation. To address the question of a possible close phylogenetic relationship between primates and Tupaia, the evolutionary affinities among primates, Tupaia and bats as representatives of the Archonta superorder, ferungulates, guinea pigs, armadillos, rats, mice, and hedgehogs were examined on the basis of the complete mitochondrial DNA sequences. The opossum sequence was used as an outgroup. The trees, estimated from 12 concatenated genes encoded on the mitochondrial H-strand, add further molecular evidence against an Archonta monophyly. With the new data described in this paper, most of both the mitochondrial and the nuclear data point away from Scandentia as the closest extant relatives to primates. Instead, the complete mitochondrial data support a clustering of Scandentia with Lagomorpha connecting to the branch leading to ferungulates. This closer phylogenetic relationship of Tupaia to rabbits than to primates first received support from several analyses of nuclear and partial mitochondrial DNA data sets. Given that short sequences are of limited use in determining deep mammalian relationships, the partial mitochondrial data available to date support this hypothesis only tentatively. Our complete mitochondrial genome data therefore add considerably more evidence in support of this hypothesis.

Nuclear integrations of mitochondrial DNA in primates: inference of associated mutational events.

To infer the possible mutational events taking place along the interorganellar transfer of genetic material from mitochondria to the nucleus, four integrations of mitochondrial DNA (mtDNA) in the human genome were characterized together with their flanking nuclear sequences. By determining their presence/absence status in different primate species, these integrations were inferred to have occurred on the lineages leading to catarrhines (Old World monkeys and hominoids), to hominoids and to humans, respectively. In case of a polymorphic state, with respect to its presence in a certain species, each preintegration sequence was either cloned in the same species or in a primate taxon that branched off before the transfer of the mtDNA to the nucleus took place. For the four mtDNA integrations presented here, random mobilization of the mtDNA and differing mechanisms for generating free ends in the nuclear target sequences can be inferred. Additionally, no common sequence features at the preintegration sites could be observed for these integrations. Moreover, the comparisons of the sites before and after integration suggest different ways of integration. Thus, mtDNA integrations represent unique molecular recombinations in the evolutionary history and can, according to their presence/absence status in different species, help to determine the branching order in phylogenetic trees.

A hominoid-specific nuclear insertion of the mitochondrial D-loop: implications for reconstructing ancestral mitochondrial sequences.

A nuclear integration of a mitochondrial control region sequence on human chromosome 9 has been isolated. PCR analyses with primers specific for the respective insertion-flanking nuclear regions showed that the insertion took place on the lineage leading to Hominoidea (gibbon, orangutan, gorilla, chimpanzee, and human) after the Old World monkey-Hominoidea split. The sequences of the control region integrations were determined for humans, chimpanzees, gorillas, orangutans, and siamangs. These sequences were then used to construct phylogenetic trees with different methods, relating them with several hominoid, Old Work monkey, and New World monkey mitochondrial control region sequences. Applying maximum-likelihood, neighbor-joining, and parsimony algorithms, the insertion clade was attached to the branch leading to the hominoid mitochondrial sequences as expected from the PCR-determined presence/absence of this integration. An unexpected long branch leading to the internal node that connects all insertion sequences was observed for the different phylogeny reconstruction procedures. This finding is not totally compatible with the lower evolutionary rate in the nucleus than in the mitochondrial compartment. We determined the unambiguous substitutions on the branch leading to the most recent common ancestor (MRCA) of the mitochondrial inserts according to the parsimony criterium. We propose that they are unlikely to have been caused by damage of the transposing nucleic acid and that they are probably due to a change in the evolutionary mode after the transposition.

Novel mitochondrial DNA insertion polymorphism and its usefulness for human population studies.

The frequency of a polymorphic mitochondrial DNA insertion into the nuclear genome was determined for 870 individuals from a geographically diverse set of 20 populations. The mtDNA insertion frequency varies significantly among populations, having a large GST value (0.178) and high heterozygosity values within populations. The clinal pattern of increasing frequency of the insertion from Africans through Europeans and Asians to native Americans is striking. The polymorphism is a new example of insertion-deletion polymorphisms and is a valuable marker for human population and evolutionary studies.

A nuclear 'fossil' of the mitochondrial D-loop and the origin of modern humans.

Mammalian mitochondrial DNA sequences evolve more rapidly than nuclear sequences. Although the rapid rate of evolution is an advantage for the study of closely related species and populations, it presents a problem in situations where related species, used as outgroups in phylogenetic analyses, have accumulated so much change that multiple substitutions obliterate the phylogenetic information. However, mitochondrial DNA sequences are frequently inserted into the nuclear genome, where they presumably evolve as nuclear pseudogene sequences and therefore more slowly than their mitochondrial counterparts. Such sequences thus represent molecular 'fossils' that could shed light on the evolution of the mitochondrial genome and could be used as outgroups in situations where no appropriate outgroup species exist. Here we show that human chromosome 11 carries a recent integration of the mitochondrial control region that can be used to gain further insight into the origin of the human mitochondrial gene pool.

Apoptosis of T lymphocytes in experimental autoimmune encephalomyelitis. Evidence for programmed cell death as a mechanism to control inflammation in the brain.

In experimental autoimmune encephalomyelitis (EAE) myelin-specific T lymphocytes attack the myelinated tissue of the central nervous system (CNS). In the Lewis rat, EAE as a rule has an acute, monophasic course. With spontaneous clinical recovery the inflammatory CNS infiltrates are cleared from the nervous tissue within a few days. This is well in line with the remarkably low incidence of myelin-specific T cells present in EAE infiltrate. Combining immunocytochemical techniques, ultrastructural criteria and in situ nick translation we found up to 49% of T lymphocytes in EAE lesions showing signs of apoptosis at the time of recovery from disease. Our results suggest that apoptosis of T lymphocytes may be one possible mechanism to eliminate T lymphocytes from inflammatory brain lesions.

Detection of DNA fragmentation in apoptosis: application of in situ nick translation to cell culture systems and tissue sections.

Since DNA fragmentation is a key feature of programmed cell death (PCD) and also occurs in certain stages of necrosis, we have adapted the methodology of in situ nick-translation (ISNT) to detect DNA fragmentation on a single-cell level. We first established the technique for cell preparations. Apoptosis was induced by gamma-irradiation on freshly isolated rat thymocytes. After fixation procedures, ISNT was performed by overnight incubation either with fluorescein-12-dUTP or with digoxigenin-labeled 11-dUTP and DNA polymerase I. The enzymatic incorporation of labeled nucleotides at sites of DNA fragmentation was detected by flow cytometry either directly or indirectly with fluorescein-conjugated anti-digoxigenin. The quantitative results demonstrated close correlation with morphological essays for apoptosis, DNA gel electrophoresis, and ISNT. Proliferating cells determined by bromodeoxyuridine immunofluorescence were not labeled by ISNT. Immunocytochemistry for cell surface antigens in combination with ISNT allowed the identification of specific cell types undergoing PCD. Furthermore, the simultaneous application of photolabeling techniques with ethidium monoazide and ISNT led to the identification of DNA fragmentation in cells with still intact membranes. Extending ISNT to tissue sections of paraformaldehyde-fixed, paraffin-embedded material reliably revealed labeling of cells with typical morphological features of apoptosis. However, this technique was not useful in detecting early stages of necrotic cell death.

Characterization of hypervariable locus-specific probes derived from a (CAC)5/(GTG)5 multilocus fingerprint in various Eurasian populations.

Population genetic studies were performed using oligonucleotide probes (Hz1103, Hz4103, and Hz4201) that recognize three hypervariable loci (D11S859, D9S128 and D22S265) in the human genome. DNA from 17 Eurasian population samples including 37 monozygotic twin pairs were digested with HinfI and hybridized with Hz4103. Allele frequency distribution profiles and high degrees of heterozygosity were similar in each ethnic group. Among 804 unrelated individuals tested, we detected one case of mosaicism caused by a somatic recombination event in a monozygotic twin. In addition, samples of DNA from three ethnic groups (Germans, Assamese Hindus and Thais) and from German and Thai families were restricted with MboI and probed with Hz1103, Hz4103, and Hz4201. The results showed considerable degrees of heterozygosity and locus-specific allele distribution profiles, rather than interpopulation differences. Among 262 meioses (12 three-generation families with a total of 131 children) analyzed, a single recombination event was observed following hybridization with the DNA probe Hz4201.

Dissecting (CAC)5/(GTG)5 multilocus fingerprints from man into individual locus-specific, hypervariable components.

Individual components of multilocus fingerprints from man produced by (CAC)5/(GTG)5 oligonucleotides have been scrutinized to characterize their peculiar properties. Successful cloning and changes occurring during the propagation of recombinant simple repetitive DNA in prokaryotic hosts are described. The isolated locus-specific probes were characterized with respect to their formal (and population genetic) properties and their usefulness for individualization and linkage studies. The localization was determined on chromosomes 8, 9, 11, and 22. Repeat flanking sequences were characterized and analyzed for their coding potential because of significant open reading frames and apparent evolutionary conservation among vertebrates. The organization of the repeats and their flanking regions in the human genome is discussed with respect to the sequence (fine) architecture that developed during evolution. Classical "minisatellite" sequences were not detected near hypervariable (cac)n/(gtg)n repeats. The single-copy probes described herein are a convenient complement to the oligonucleotides employed for multilocus fingerprinting. Many practical applications are apparent.

Oligonucleotide fingerprinting to individualize ungulates.

The optimal combination of restriction enzyme and oligonucleotide probe has been determined for the individualization of hoofed animal species (cattle, pig, goat, sheep, horse and camel). Four different restriction endonucleases were used as well as five synthetic oligonucleotide probes hybridizing to different simple tandem repeats for fingerprint analyses in unrelated cattle (Swiss and German Simmental of unknown relationship and three families): 4 x 10(15) cows and oxes would reveal different banding patterns after HinfI digestion using the probe (CAC)5/(GTG)5. The other species were investigated using HinfI (and HaeIII) and five different oligonucleotide probes specific for simple tandem repeats. Using (CAC)5/(GTG)5 the discrimination potential in sheep was about one order of magnitude lower than in cattle while in goats 6 x 10(10) specimen are easily differentiated with (CA)8/(GT)8. From an evolutionary standpoint it may be of interest that also in all other ungulate species tested, (CAC)5/(GTG)5 and (CA)8/(GT)8 exhibited the highest potential for individualization. Advantages of oligonucleotide fingerprinting are discussed.

Coamplification of simple repetitive DNA fingerprint fragments and the EGFR gene in human gliomas.

DNA fingerprints were generated by the oligonucleotide probe (GTG)5 from surgically removed tissue and/or primary cell culture of 36 intracranial tumors (31 gliomas, 1 medulloblastoma, 4 metastatic carcinomas) and compared with the constitutional banding pattern obtained from the peripheral blood leukocytes of each patient. A multitude of somatic changes was detected and found to reflect the chromosome alterations identified by parallel karyotype analysis. Gain and/or loss of bands or significant band intensity shifts could be demonstrated in the fingerprints of more than 80% of the tumors investigated. This included a highly amplified fingerprint fragment in five independent gliomas (four of them had double minutes, dmin) which appeared not individual- but tumor-specific (2.4 kilobases, kb, after HaeIII digestion). Rehybridization with the oligonucleotide probes (GT)8 and (GATA)4, respectively, revealed additional amplified fingerprint fragments in the tumor DNA of these patients. While a (ca/gt)n fragment (2.6 kb. HaeIII) was also found to be amplified in all five cases, one band detected with (GATA)4 (1.4 kb, HaeIII) represented a unique feature for one of these tumors only. Amplification of the epidermal growth factor receptor (EGFR) gene via Southern blot hybridization was revealed only in those tumors showing the amplified DNA fingerprint fragments as well. Thus in many gliomas the amplification unit harbors two simple repetitive DNA fingerprint loci, (cac/gtg)n and (ca/gt)n, in addition to the EGFR gene.

Oligonucleotide fingerprinting with (CAC)5: nonradioactive in-gel hybridization and isolation of individual hypervariable loci.

The first topic to be treated in this paper is the nonradioactive DNA fingerprinting by means of in-gel hybridization with digoxigenated (CAC)5. Besides the fact that time-consuming Southern blotting can be avoided, the dried agarose is an excellent matrix to produce background-free nonradioactive DNA fingerprints. There is no tendency of either the oligonucleotide probe or the antibody towards unspecific binding to the dried agarose. Prehybridization and blocking steps are therefore superfluous. Furthermore, we will discuss what effect the degree of crosslinking of the antibody-enzyme conjugates has. The second topic concerns the isolation and characterization of locus-specific probes from a human (CAC)5 fingerprint. The isolation and characterization of one variable probe, by screening complete genomic libraries, is described and discussed. This probe is compared to a hypervariable single-copy probe, isolated from a size-enriched genomic library. The sequence of the repeat flanking locus-specific probe is presented and a semi-specific, adaptor-mediated polymerase chain reaction was designed to amplify (CAC)n/(GTG)n flanking sequences.