Ivan Alexandrov

The phylogeny of human chromosome specific alpha satellites.

The chromosomal distribution of sequences homologous to 18 coned alpha satellite fragments was established by in situ hybridization. It appeared that all the cloned sequences were members of small repeated families located on single chromosome pairs. Among the sequences studied specific molecular markers for chromosomes 3, 4, 10,11,17,18 and X were found. Comparison of the hybridization spectra obtained under non-stringent conditions and of restriction site periodicities in different chromosome-specific families allowed the identification of three “suprachromosomal” families, each located on a characteristic set of chromosomes. The three families together cover all the autosomes and the X chromosome. These data plus those reported previously allow part of the phylogenetic tree of chromosome-specific alpha satellite repeats to be drawn. Each suprachromosomal family has presumably originated from a distinct ancestral sequence and consists of certain types of monomers. Ancestral sequences have evolved into a number of chromosome-specific families by cycles of interchromosomal transfers and subsequent amplification events. The high homogeneity of chromosome-specific families may be a result of intrachromosomal homogenization of amplification units in chromosome-specific alpha satellite domains.

Chromosome-specific alpha satellites: Two distinct families on human chromosome 18

Two types of human chromosome 18-specific alpha satellite fragments have been cloned and sequenced. They represent closely related but distinct alphoid families formed by two different types of the higher-order repeated units (1360-bp EcoRI and 1700-bp HindIII fragments) that do not alternate in the genome. The individual repeats within each family are 99% identical and interfamily homology is about 78%. Sequence analysis shows that both repeats belong to alphoid suprachromosomal family 2, but their homology is not higher than that of family members located on different chromosomes. Therefore, the two repeats shared a common origin in the recent past, although they are not the direct offspring of one ancestral sequence. Our data indicate that these two 18-specific domains have appeared as a result of two separate amplification events. Despite the high degree of homology, they are not undergoing intrachromosomal homogenization, although some variation of this process might take place within each domain.

Ap4A induces apoptosis in human cultured cells

Diadenosine oligophosphates (ApnA) have been proposed as intracellular and extracellular signaling molecules in animal cells. The ratio of diadenosine 5′,5‴‐P1,P3‐triphosphate to diadenosine 5′,5‴‐P1,P4‐tetraphosphate (Ap3A/Ap4A) is sensitive to the cellular status and alters when cultured cells undergo differentiation or are treated with interferons. In cells undergoing apoptosis induced by DNA topoisomerase II inhibitor VP16, the concentration of Ap3A decreases significantly while that of Ap4A increases. Here, we have examined the effects of exogenously added Ap3A and Ap4A on apoptosis and morphological differentiation. Penetration of ApnA into cells was achieved by cold shock. Ap4A at 10 μM induced programmed cell death in human HL60, U937 and Jurkat cells and mouse VMRO cells and this effect appeared to require Ap4A breakdown as hydrolysis‐resistant analogues of Ap4A were inactive. On its own, Ap3A induced neither apoptosis nor cell differentiation but did display strong synergism with the protein kinase C activators 12‐deoxyphorbol‐13‐O‐phenylacetate and 12‐deoxyphorbol‐13‐O‐phenylacetate‐20‐acetate in inducing differentiation of HL60 cells. We propose that Ap4A and Ap3A are physiological antagonists in determination of the cellular status: Ap4A induces apoptosis whereas Ap3A is a co‐inductor of differentiation. In both cases, the mechanism of signal transduction remains unknown.

Application of cloned satellite DNA sequences to molecular-cytogenetic analysis of constitutive heterochromatin heteromorphisms in man

SummaryThe cloned alpha-satellite DNA sequences were used to evaluate the specificity and possible variability of repetitive DNA in constitutive heterochromatin of human chromosomes. Five probes with high specificity to individual chromosomes (chromosomes 3, 11, 17, 18, and X) were in situ hybridized to metaphase chromosomes of different individuals. The stable position of alpha-satellite DNA sequences in heterochromatic regions of particular chromosomes was found. Therefore, the chromosome-specific alpha-satellite DNA sequences may be used as molecular markers for heterochromatic regions of certain human chromosomes. The homologous chromosomes of many individuals were characterized by cytologically visible heteromorphisms of hybridization intensity with chromosome-specific alpha-satellite DNA sequences. A special analysis of hybridization between homologues with morphological differences provided the evidence for a high resolution power of the in situ hybridization technique for evaluation of chromosome heteromorphisms. The approaches for detection of heteromorphisms in cases without morphological differences between homologues are discussed. The results obtained indicate that constitutive heterochromatin of human chromosomes has a variable amount of alphasatellite DNA sequences. In situ hybridization of cloned satellite DNA sequences may be used as a new general approach to analysis of chromosome heteromorphisms in man.

The Evolutionary Origin of Man Can Be Traced in the Layers of Defunct Ancestral Alpha Satellites Flanking the Active Centromeres of Human Chromosomes

Alpha satellite domains that currently function as centromeres of human chromosomes are flanked by layers of older alpha satellite, thought to contain dead centromeres of primate progenitors, which lost their function and the ability to homogenize satellite repeats, upon appearance of a new centromere. Using cladistic analysis of alpha satellite monomers, we elucidated complete layer patterns on chromosomes 8, 17, and X and related them to each other and to primate alpha satellites. We show that discrete and chronologically ordered alpha satellite layers are partially symmetrical around an active centromere and their succession is partially shared in non-homologous chromosomes. The layer structure forms a visual representation of the human evolutionary lineage with layers corresponding to ancestors of living primates and to entirely fossil taxa. Surprisingly, phylogenetic comparisons suggest that alpha satellite arrays went through periods of unusual hypermutability after they became “dead” centromeres. The layer structure supports a model of centromere evolution where new variants of a satellite repeat expanded periodically in the genome by rounds of inter-chromosomal transfer/amplification. Each wave of expansion covered all or many chromosomes and corresponded to a new primate taxon. Complete elucidation of the alpha satellite phylogenetic record would give a unique opportunity to number and locate the positions of major extinct taxa in relation to human ancestors shared with extant primates. If applicable to other satellites in non-primate taxa, analysis of centromeric layers could become an invaluable tool for phylogenetic studies.

Unequal cross‐over is involved in human alpha satellite DNA rearrangements on a border of the satellite domain

It can be invoked from the theory of tandem repeat homogenization that DNA on a satellite/non‐satellite border may carry sequence marks of molecular processes basic to satellite evolution. We have sequenced a continuous 17‐kb alpha satellite fragment bordering the non‐satellite in human chromosome 21, which is devoid of higher‐order repeated structure, contains multiple rearrangements, and exhibits higher divergence of monomers towards the border, indicating the lack of efficient homogenization. Remarkably, monomers have been found with mutually supplementary deletions matching each other as reciprocal products of unequal recombination, which provide evidence for unequal cross‐over as a mechanism generating deletions in satellite DNA.

Interspersed repeats are found predominantly in the “old” α satellite families

The biased distribution of dispersed repeat insertions in various types of primate specific α satellites (AS) is being discussed in the literature in relation to the modes of AS evolution and their possible roles in maintenance and disruption of functional centromeres. However, such a bias has not been properly documented on a genome-wide scale so far. In this work, using a representative sample of about 100 insertions we show that the “old” AS contains at least 10 times more dispersed repeats than the “new” one. In the new arrays insertions accumulate mostly in poorly homogenized areas, presumably in the edges, and in the old AS, throughout the whole array length. Dating of L1 insertions in the old AS revealed that their massive accumulation started at or after the time when the new AS emerged and expanded in the genome and the centromere function had shifted to the new AS arrays.

Annotation of suprachromosomal families reveals uncommon types of alpha satellite organization in pericentromeric regions of hg38 human genome assembly

Centromeric alpha satellite (AS) is composed of highly identical higher-order DNA repetitive sequences, which make the standard assembly process impossible. Because of this the AS repeats were severely underrepresented in previous versions of the human genome assembly showing large centromeric gaps. The latest hg38 assembly (GCA_000001405.15) employed a novel method of approximate representation of these sequences using AS reference models to fill the gaps. Therefore, a lot more of assembled AS became available for genomic analysis. We used the PERCON program previously described by us to annotate various suprachromosomal families (SFs) of AS in the hg38 assembly and presented the results of our primary analysis as an easy-to-read track for the UCSC Genome Browser. The monomeric classes, characteristic of the five known SFs, were color-coded, which allowed quick visual assessment of AS composition in whole multi-megabase centromeres down to each individual AS monomer. Such comprehensive annotation of AS in the human genome assembly was performed for the first time. It showed the expected prevalence of the known major types of AS organization characteristic of the five established SFs. Also, some less common types of AS arrays were identified, such as pure R2 domains in SF5, apparent J/R and D/R mixes in SF1 and SF2, and several different SF4 higher-order repeats among reference models and in regular contigs. No new SFs or large unclassed AS domains were discovered. The dataset reveals the architecture of human centromeres and allows classification of AS sequence reads by alignment to the annotated hg38 assembly. The data were deposited here: http://genome.ucsc.edu/cgi-bin/hgTracks?db=hg38&hgt.customText=https://dl.dropboxusercontent.com/u/22994534/AS-tracks/human-GRC-hg38-M1SFs.bed.bz2.

c-Raf kinase binds to N-terminal domain of c-Myc

We have demonstrated that the 50 N‐terminal amino acids of c‐Myc bind a kinase activity, which phosphorylates Myc in vitro predominantly on Thr8. We also have shown that c‐Raf, a widely known Ser/Thr kinase, involved in the Ras signaling pathway, binds to the same portion of c‐Myc in vitro. In addition we were able to precipitate native c‐Myc/Raf complex from various cell lysates. Physical interaction of Myc and Raf may potentially be a part of their well‐known functional cooperation.

Classification and monomer-by-monomer annotation of suprachromosomal family 1 alpha satellite higher-order repeats in hg38 human genome assembly

In the latest hg38 human genome assembly, centromeric gaps has been filled in by alpha satellite (AS) reference models (RMs) which are statistical representations of homogeneous higher-order repeat (HOR) arrays that make up the bulk of the centromeric regions. We studied these models to compose an atlas of human HORs where each monomer of a HOR could be characterized and represented by a number of its polymorphic sequence variants. We further used these data and HMMER sequence analysis platform to annotate AS HORs in the assembly. This led to discovery and annotation of a new type of low copy number highly divergent HORs which were not represented by RMs. The annotation can be viewed as UCSC Genome Browser custom track (the HOR-track) and used together with our previous annotation of AS SFs in the same assembly where each AS monomer can be viewed in its genomic context together with its classification into one of the 5 major SFs (the SF-track). To catalog the diversity of AS HORs in the human genome we introduced a new naming system. Each HOR received a name which showed its SF, chromosomal location and index number. Here we present the first installment of the HOR-track covering only the 17 HORs that belong to SF1 which forms live functional centromeres in chromosomes 1, 3, 5, 6, 7, 10, 12, 16 and 19 and also a large number of minor dead HOR domains, both homogeneous (pseudo) and divergent (relic). The 4 newly discovered divergent SF1 HORs have provided the missing links in SF1 early evolution and substantiated its partition into 2 generations, archaic and modern, which we reported earlier. Additionally, we demonstrated that monomer-by-monomer HOR annotation was useful for mapping and quantification of various structural variants of AS HORs which would be important for studies of inter-individual polymorphism of AS including centromeric functional epialleles.