Occurrence of MIR Elements in the Complete Nucleotide Sequence of Human Chromosome 22

The location of mammalian interspersed repeats (MIRs) and their density have been determined in the complete nucleotide sequence of human chromosome 22. The approach developed by us has allowed detection of 9675 MIRs at a statistically significant level, which by 15% exceeds the MIR number revealed by all previous approaches. It has been demonstrated that a considerable amount of MIRs missed by the algorithms applied earlier occurs in known DNA sequences of the human genome. The study of the MIR density revealed substantial irregularity of their distribution along the chromosome. The data on the MIRs thus found and the computer program searching for diverged sequences are available by E-mail: katrin2@mail.ru or katrin22@mtu-net.ru.     

Evolution of MIR-repeats in coding regions of the human genome]

A search for new members of the mammalian interspersed repeat (MIR) family has been done over the coding regions of human genome from GenBank-116. Only 254 nucleotide sequences contained MIRs in coding regions, of which 45 MIR copies were unknown before, including 17 that occurred in translated gene regions. The program developed by the authors has been demonstrated to surpass the CENSOR program in the search power. The evolution of the MIR copies located in translated regions of human genome is discussed.     

Evolution of MIR Elements Located in the Coding Regions of Human Genome

A search for new members of the mammalian interspersed repeat (MIR) family has been done over the coding regions of human genome from GenBank-116. Only 254 nucleotide sequences contained MIRs in coding regions, of which 45 MIR copies were unknown before, including 17 that occurred in translated gene regions. The program developed by the authors has been demonstrated to surpass the CENSOR program in the search power. The evolution of the MIR copies located in translated regions of human genome is discussed.     

MIR: Family of repeats common to vertebrate genomes

We studied the occurrence of mammalian interspersed repeats (MIRs) in DNA and RNA of vertebrates, invertebrates, and bacteria using the data from GenBank. A special algorithm based on a weight position matrix with optimal alignment using dynamic programming was developed to search for the traces of MIR dissemination. This allowed us to search for highly divergent MIRs carrying deletions and insertions. MIRs were detected in genomes of various fishes, includingLatimeria. This suggests that the origin of MIRs dates back more than 400 million years. The method to search for similarity between highly divergent sequences may be used to find the genome fragments from various ancient repeat families and from various gene families.     

Evolutionary implications of multiple SINE insertions in an intronic region from diverse mammals

An analysis of the nuclear β-fibrinogen intron 7 locus from 30 taxa representing 12 placental orders of mammals reveals the enriched occurrences of short interspersed element (SINE) insertion events. Mammalian-wide interspersed repeats (MIRs) are present at orthologous sites of all examined species except those in the order Rodentia. The higher substitution rate in mouse and a rare MIR deletion from rat account for the absence of MIR in the rodents. A minimum of five lineage-specific SINE sequences are also found to have independently inserted into this intron in Carnivora, Artiodactyla and Lagomorpha. In the case of Carnivora, the unique amplification pattern of order-specific CAN SINE provides important evidence for the “pan-carnivore” hypothesis of this repeat element and reveals that the CAN SINE family may still be active today. Particularly interesting is the finding that all identified lineage-specific SINE elements show a strong tendency to insert within or in very close proximity to the preexisting MIRs for their efficient integrations, suggesting that the MIR element is a hot spot for successive insertions of other SINEs. The unexpected MIR excision as a result of a random deletion in the rat intron locus and the non-random site targeting detected by this study indicate that SINEs actually have a greater insertional flexibility and regional specificity than had previously been recognized. Implications for SINE sequence evolution upon and following integration, as well as the fascinating interactions between retroposons and the host genomes are discussed.     

Characterization of functional transposable element enhancers in acute myeloid leukemia

Transposable elements(TEs) have been shown to have important gene regulatory functions and their alteration could lead to disease phenotypes. Acute myeloid leukemia(AML) develops as a consequence of a series of genetic changes in hematopoietic precursor cells, including mutations in epigenetic factors. Here, we set out to study the gene regulatory role of TEs in AML. We first explored the epigenetic landscape of TEs in AML patients using ATAC-seq data. We show that a large number of TEs in general, and more specifically mammalian-wide interspersed repeats(MIRs), are more enriched in AML cells than in normal blood cells. We obtained a similar finding when analyzing histone modification data in AML patients. Gene Ontology enrichment analysis showed that genes near MIRs in open chromatin regions are involved in leukemogenesis. To functionally validate their regulatory role, we selected 19 MIR regions in AML cells, and tested them for enhancer activity in an AML cell line(Kasumi-1) and a chronic myeloid leukemia(CML) cell line(K562); the results revealed several MIRs to be functional enhancers. Taken together, our results suggest that TEs are potentially involved in myeloid leukemogenesis and highlight these sequences as potential candidates harboring AML-associated variation.     

Structural organization of the human genome: distribution of nucleotides, Alu-repeats and exons in chromosomes 21 and 22]

Analysis of DNA sequences of the human chromosomes 21 and 22 performed using a specially designed MegaGene software allowed us to obtain the following results. Purine and pyrimidine nucleotide residues are unevenly distributed along both chromosomes, displaying maxima and minima (Y waves phi) with a period of about 3 Mbp. Distribution of G + C along both chromosomes has no distinct maxima and minima, however, chromosome 21 contains considerably less G + C than chromosome 22. Both exons and Alu repeats are unevenly distributed along chromosome 21: they are scarce in its left part and abundant in the right part, while MIR elements are quite monotonously spread along this chromosome. The Alu repeats show a wave-like distribution pattern similar for both repeat orientations. The number of the Alu repeats of opposite orientations was equal for both studied chromosomes, and this may be considered a new property of the human genome. The positive correlation between the exon and Alu distribution patterns along the chromosome, the concurrent distribution of Alu repeats in both orientations along the chromosome, and the equal copy numbers for Alu in direct and inverted orientations within an individual chromosome point to their important role in the human genome, and do not fit the notion that Alu repeats belong to parasitic (junk) DNA.     

Transposable elements in disease-associated cryptic exons

Transposable elements (TEs) make up a half of the human genome, but the extent of their contribution to cryptic exon activation that results in genetic disease is unknown. Here, a comprehensive survey of 78 mutation-induced cryptic exons previously identified in 51 disease genes revealed the presence of TEs in 40 cases (51%). Most TE-containing exons were derived from short interspersed nuclear elements (SINEs), with Alus and mammalian interspersed repeats (MIRs) covering >18 and >16% of the exonized sequences, respectively. The majority of SINE-derived cryptic exons had splice sites at the same positions of the Alu/MIR consensus as existing SINE exons and their inclusion in the mRNA was facilitated by phylogenetically conserved changes that improved both traditional and auxiliary splicing signals, thus marking intronic TEs amenable for pathogenic exonization. The overrepresentation of MIRs among TE exons is likely to result from their high average exon inclusion levels, which reflect their strong splice sites, a lack of splicing silencers and a high density of enhancers, particularly (G)AA(G) motifs. These elements were markedly depleted in antisense Alu exons, had the most prominent position on the exon–intron gradient scale and are proposed to promote exon definition through enhanced tertiary RNA interactions involving unpaired (di)adenosines. The identification of common mechanisms by which the most dynamic parts of the genome contribute both to new exon creation and genetic disease will facilitate detection of intronic mutations and the development of computational tools that predict TE hot-spots of cryptic exon activation.     

Tandem Fusion of Hepatitis B Core Antigen Allows Assembly of Virus-Like Particles in Bacteria and Plants with Enhanced Capacity to Accommodate Foreign Proteins

The core protein of the hepatitis B virus, HBcAg, assembles into highly immunogenic virus-like particles (HBc VLPs) when expressed in a variety of heterologous systems. Specifically, the major insertion region (MIR) on the HBcAg protein allows the insertion of foreign sequences, which are then exposed on the tips of surface spike structures on the outside of the assembled particle. Here, we present a novel strategy which aids the display of whole proteins on the surface of HBc particles. This strategy, named tandem core, is based on the production of the HBcAg dimer as a single polypeptide chain by tandem fusion of two HBcAg open reading frames. This allows the insertion of large heterologous sequences in only one of the two MIRs in each spike, without compromising VLP formation. We present the use of tandem core technology in both plant and bacterial expression systems. The results show that tandem core particles can be produced with unmodified MIRs, or with one MIR in each tandem dimer modified to contain the entire sequence of GFP or of a camelid nanobody. Both inserted proteins are correctly folded and the nanobody fused to the surface of the tandem core particle (which we name tandibody) retains the ability to bind to its cognate antigen. This technology paves the way for the display of natively folded proteins on the surface of HBc particles either through direct fusion or through non-covalent attachment via a nanobody.     

Contribution to the Prediction of the Fold Code: Application to Immunoglobulin and Flavodoxin Cases

Background

Folding nucleus of globular proteins formation starts by the mutual interaction of a group of hydrophobic amino acids whose close contacts allow subsequent formation and stability of the 3D structure. These early steps can be predicted by simulation of the folding process through a Monte Carlo (MC) coarse grain model in a discrete space. We previously defined MIRs (Most Interacting Residues), as the set of residues presenting a large number of non-covalent neighbour interactions during such simulation. MIRs are good candidates to define the minimal number of residues giving rise to a given fold instead of another one, although their proportion is rather high, typically [15-20]% of the sequences. Having in mind experiments with two sequences of very high levels of sequence identity (up to 90%) but different folds, we combined the MIR method, which takes sequence as single input, with the “fuzzy oil drop” (FOD) model that requires a 3D structure, in order to estimate the residues coding for the fold. FOD assumes that a globular protein follows an idealised 3D Gaussian distribution of hydrophobicity density, with the maximum in the centre and minima at the surface of the “drop”. If the actual local density of hydrophobicity around a given amino acid is as high as the ideal one, then this amino acid is assigned to the core of the globular protein, and it is assumed to follow the FOD model. Therefore one obtains a distribution of the amino acids of a protein according to their agreement or rejection with the FOD model.

Results

We compared and combined MIR and FOD methods to define the minimal nucleus, or keystone, of two populated folds: immunoglobulin-like (Ig) and flavodoxins (Flav). The combination of these two approaches defines some positions both predicted as a MIR and assigned as accordant with the FOD model. It is shown here that for these two folds, the intersection of the predicted sets of residues significantly differs from random selection. It reduces the number of selected residues by each individual method and allows a reasonable agreement with experimentally determined key residues coding for the particular fold. In addition, the intersection of the two methods significantly increases the specificity of the prediction, providing a robust set of residues that constitute the folding nucleus.     

The Genetic and Cytogenetic Localization of the Three Structural Genes Coding for the Major Protein of Drosophila Larval Serum

The alpha, beta and gamma polypeptides that make up Drosophila Larval Serum Protein-1 seem to be coded for by genes that have evolved by duplication of a common ancestral gene. We have found variants of all three polypeptides, and these are variants of the coding sequences. The alpha-chain variant mapped to 39.5 on the X chromosome and to the polytene interval 11A7-11B9. The beta-chain variant mapped to 1.9 on chromosome 2L and to 21D2-22A1. The gamma-chain variant was mapped as 0.13 map units from the tip of chromosome 3L or to --1.41 with respect to ru, which has been defined as 0.0, and to 61A1-61A6.     

Rapid isolation of cosmids from defined subregions by differential Alu-PCR hybridization on chromosome 22-specific library.

A method based on the differential screening of a chromosome-specific cosmid library with amplified inter-Alu sequences obtained from a set of somatic cell hybrids has been developed to target the isolation of probes from predefined subchromosomal regions. As a model system, we have used a chromosome 22-specific cosmid library and four cell hybrids containing different parts of this chromosome. The procedure has identified cosmids that demonstrate differential hybridization signals with Alu-PCR products from these cell hybrids. We show, by in situ hybridization or individual mapping, that their hybridization pattern is indicative of their sublocalization on chromosome 22, thus resulting in a large enrichment factor for the isolation of probes from specific small chromosome subregions. Depending on the local Alu-sequence density, from 3 to 10 independent loci per megabase of genome can thus be identified.     

Structural Organization of the Human Genome: Distribution of Nucleotides,AluRepeats, and Exons in Chromosomes 21 and 22

Analysis of DNA sequences of the human chromosomes 21 and 22 performed using a specially designed MegaGene software allowed us to obtain the following results. Purine and pyrimidine nucleotide residues are unevenly distributed along both chromosomes, displaying maxima and minima (waves) with a period of about 3 Mbp. Distribution of G+C along both chromosomes has no distinct maxima and minima, however, chromosome 21 contains considerably less G+C than chromosome 22. Both exons and Alurepeats are unevenly distributed along chromosome 21: they are scarce in its left part and abundant in the right part, while MIR elements are quite monotonously spread along this chromosome. The Alurepeats show a wave-like distribution pattern similar for both repeat orientations. The number of the Alurepeats of opposite orientations was equal for both studied chromosomes, and this may be considered a new property of the human genome. The positive correlation between the exon and Aludistribution patterns along the chromosome, the concurrent distribution of Alurepeats in both orientations along the chromosome, and the equal copy numbers for Aluin direct and inverted orientations within an individual chromosome point to their important role in the human genome, and do not fit the notion that Alurepeats belong to parasitic (junk) DNA.     

Interstitial telomere repeats as markers of evolutionary changes in the mammalian karyotype: human chromosome 2

Telomer repeats represented by hexamer (TTAGGG)n at chromosome termini are required for correct function and chromosome stability. At the same time, interstitial telomer sequence (ITS) located far from the chromosome ends are known for several mammalian genomes, including the human genome. It is assumed that these repeats mark the points of fusion or other chromosome reconstructions of ancestors. Exact localization of all interstitial telomer sequences in the genome could greatly improve our understanding of the mechanism of karyotype evolution and species origin. We have developed a software for a search of interstitial telomer sequences in complete sequences of mammalian genomes. We have demonstrated the evolutionary significance of repeats by an example of human chromosome 2. The results and supplementary materials are available at the site of the Institute of Cytology and Genetics: http://www.bionet.nsc.ru/labs/theorylabmain/orlov/telomere/.     

Isolation and regional mapping of DNA sequences unique to human chromosome 21.

To isolate DNA sequences unique to chromosome 21 we have used a recombinant-DNA library, constructed from a mouse-human somatic-cell hybrid line containing chromosome 21 as the only human chromosome. Individual recombinant phage containing human DNA inserts were identified by their hybridization to total human DNA sequences and by their failure to hybridize to total mouse DNA sequences. A repeat-free human DNA fragment was then subcloned from each of 14 such recombinant phage. An independent somatic-cell hybrid was used to assign all 14 subcloned fragments to chromosome 21. Thirteen of the fragments have been regionally mapped using a somatic-cell hybrid containing a human 21 translocation chromosome. Two probes map proximal to the 21q21.2 translocation breakpoint, and 11 probes map distal to this breakpoint, placing them in the region 21q21.2-21q22. One of seven probes used to screen for restriction-fragment-length polymorphisms recognized polymorphic DNA fragments when hybridized to genomic DNA from unrelated individuals. These 14 unique probes provide useful tools for studying the structure and function of human chromosome 21 as well as for investigating the molecular biology of Down syndrome.     

NotI-STS markers for human chromosome 3 are gene markers

Ninety four NotI-STS markers to seventy two individual NotI clones were developed basing on DNA nucleotide sequences from NotI-"jumping" and "linking" NotI-libraries of human chromosome 3. The localization of NotI-STS markers and their ordering on chromosome was established by combined data of RH-mapping (our data), contig-mapping, cytogenetic mapping and in silico mapping. Performed comparison of NotI-STS DNAs with human genome sequences revealed two gaps in the regions, 3p21.33 (marker NLI-256) and 3p21.31 (NL3-005), and segmental duplication. Identical DNA fragments are localized in the regions 12q and 3p22-21.33 (marker NL3-007). In the region 3q28-q29 (marker NLM-084) a fragment was detected with its identical copies present also on chromosomes 1, 2, 15 and 19. For 69 NotI-STSs, significant homologies with nucleotide sequences of 70 genes and two cDNAs were detected taking in consideration homologies to NotI-STS 5'- and 3'-terminal sequences. Association of NotI-STSs with genes is confirmed by high correlation of gene density distribution with the density of NotI-STS markers on the map of human chromosome 3. Obtained data evidence possibility of NotI-STS marker application as gene markers and allow considering constructed NotI-map as gene map of human chromosome 3.     

Generation of a chromosome-22-specific c-DNA library as confirmed by FISH analysis

We have recently developed a strategy for the rapid enrichment of c-DNA fragments from selected human chromosomes. Heteronuclear RNA (hn-RNA) is isolated from a somatic cell hybrid that retains a single human chromosome in a rodent background. Following c-DNA synthesis, human sequences are selectively amplified by the Alu polymerase chain reaction (Alu-PCR). Here we have applied this protocol for the selective isolation of novel c-DNAs encoded by chromosome 22. Fluorescence in situ hybridization has been used to confirm the chromosome-22-specific origin of the c-DNA fragments. Controls show DNAse-free RNase-treated hn-RNA results in no c-DNAs or Alu-PCR products. As demonstrated by competitive in situ suppression hybridization (CISS), the majority of the Alu-PCR products from hybrid GM 10027 are located on chromosome 22. Without competition, hybridization signals have also been identified on other human chromosomes. These unspecific hybridization signals result from Alu sequences and can successfully be reduced by competition with cot 1 DNA. This is the first report of the use of CISS for the localization of chromosome-specific c-DNAs.     

Human periplakin: genomic organization in a clonally unstable region of chromosome 16p with an abundance of repetitive sequence elements

Periplakin, a member of the plakin family of proteins, has been recently characterized by cDNA cloning, and the corresponding gene, PPL, has been mapped to human chromosome 16p13.3 (Aho et al., 1998, Genomics 48: 242-247). Periplakin has also been shown to serve as an autoantigen in a malignancy-associated autoimmune blistering disease, paraneoplastic pemphigus (Mahoney et al., 1998, J. Invest. Dermatol. 111: 308-313). In this study, we have elucidated the intron-exon organization of human PPL and characterized its promoter region. The flanking 5' sequences were rich in G and C ( approximately 80%) and included multiple AP2 sites and a SP1 site, while no canonical TATA or CCAAT sequences were found. The functionality of the upstream sequences (-709 to +135) as a promoter in cultured epidermal keratinocytes was detected by a CAT reporter gene, and a limited region (-382 to +135) showed activity in cultured dermal fibroblasts, attesting to cell-type specificity of the promoter. The genomic organization, including the intron-exon borders, was determined by direct nucleotide sequencing of human genomic P1 clones. Comparative analysis of cDNA and genomic sequences revealed that PPL consists of 22 exons, with the distribution of exons in PPL being consistent with that of other plakin genes: 21 small exons, separated by large introns, encode the amino-terminal globular domain, and 1 large exon encodes the entire rod and the tail domains. Characterization of four P1 clones spanning the PPL locus revealed multiple Alu repeats, 20 of them within 33 kb of the entirely sequenced segments (0.60/kb), in addition to numerous MIR and L1 elements. These repetitive elements could lead to the clonal instability detected throughout the genomic P1 clones and may give rise to the genomic rearrangements possibly underlying the paraneoplastic pemphigus.     

Molecular analysis of both translocation products of a Philadelphia-positive CML patient.

The breakpoint regions of both translocation products of the (9;22) Philadelphia translocation of CML patient 83-H84 and their normal chromosome 9 and 22 counterparts have been cloned and analysed. Southern blotting with bcr probes and DNA sequencing revealed that the breaks on chromosome 22 occurred 3' of bcr exon b3 and that the 88 nucleotides between the breakpoints in the chromosome 22 bcr region were deleted. Besides this small deletion of chromosome 22 sequences a large deletion of chromosome 9 sequences (greater than 70 kb) was observed. The chromosome 9 sequences remaining on the 9q+ chromosome (9q+ breakpoint) are located at least 100 kb upstream of the v-abl homologous c-abl exons whereas the translocated chromosome 9 sequences (22q-breakpoint) could be mapped 30 kb upstream of these c-abl sequences. The breakpoints were situated in Alu-repetitive sequences either on chromosome 22 or on chromosome 9, strengthening the hypothesis that Alu-repetitive sequences can be hot spots for recombination.