Tamara D. Mashkova

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.

The primary structure of maize and tobacco 5 S rRNA

5 S ribosomal RNA structures from Zea mays and tobacco have been determined with two independent methods. The sequence of corn and tobacco 5 S rRNAs are identical to those of the Gramineae and the Solanaceae, respectively. There is no general structure conservation of 5 S rRNA in higher plants.

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.

Higher plant 5S rRNAs share common secondary and tertiary structure. A new three domains model.

A new model of secondary and tertiary structure of higher plant 5S RNA is proposed. It consists of three helical domains: domain alpha includes stem I; domain beta contains stems II and III and loops B and C; domain gamma consists of stems IV and V and loops D and E. Except for, presumably, a canonical RNA-A like domain alpha, the two remaining domains apparently adopt a perturbed RNA-A structure due to irregularities within internal loops B and E and three bulges occurring in the model. Bending of RNA could bring loops B and E and/or C and D closer making tertiary interactions likely. The model differs from that suggested for eukaryotic 5S rRNA, by organization of domain gamma. Our model is based on the results of partial digestion obtained with single- and double-strand RNA specific nucleases. The proposed secondary structure is strongly supported by the observation that crude plant 5S rRNA contains abundant RNA, identified as domain gamma of 5S rRNA. Presumably it is excised from the 5S rRNA molecule by a specific nuclease present in lupin seeds. Experimental results were confirmed by computer-aided secondary structure prediction analysis of all higher plant 5S rRNAs. Differences observed between earlier proposed models and our proposition are discussed.

Transcription of TIMP3, DAPK1, and AKR1B10 in squamous-cell lung cancer

Lung cancer is among the most common neoplasms in Russia, the United States, and in Western Europe and is accompanied by changes in the functional activity of many genes. The transcription levels of TIMP3, DAPK1, and AKR1B10 were compared for normal and tumor lung tissues of patients with squamous-cell cancer (SCC) by RT-PCR. A substantial increase in AKR1B10 transcription level was observed in 80% of the tumors. The transcription levels of TIMP3 and DAPK1 were significantly decreased in 76 and 72% of the tumors, respectively. The results implicated the genes in carcinogenesis in SCC, AKR1B10 acting as a potential oncogene, and TIMP3 and DAPK1 acting as potential tumor suppressor genes. It was assumed that dramatic changes in their transcription levels could be used for early diagnosis of SCC.

Genomic organization, sequence and polymorphism of the human chromosome 4-specific a-satellite DNA

Two alpha-satellite fragments specific for human chromosome 4 have been cloned and characterized. Under stringent annealing conditions, they hybridized in situ only to the pericentromeric region of chromosome 4, but under non-stringent conditions they hybridized to all chromosomes containing the sequences of alpha-satellite suprachromosomal family 2 (viz., chromosomes 2, 4, 8, 9, 13, 14, 15, 18, 20, 21 and 22). Southern blot analysis reveals the 3.2-kb higher-order repeated unit which exists in two forms: as a single MspI fragment or a combination of the 2.6-kb and 0.6-kb MspI fragments. The two chromosome-4-specific cloned sequences appear to be different parts of this repeated unit. Taken together they constitute about 60% of its length. The primary structure of the higher-order repeated unit is characterized by a dimeric periodicity of the D1-D2 type which is usual to suprachromosomal family 2. At least in one site this regularity is disrupted by monomer deletion leading to the D2-D2 monomeric order. The most likely mechanism of this monomer excision is homologous unequal crossing-over. These sequences may serve as both cytogenetic and restriction-fragment length polymorphism (RFLP) markers for the pericentromeric region of chromosome 4.

Molecular evolution of plants as deduced from changes in free energy of 5S ribosomal RNAs

The nucleotide sequence of Pinus silvestyris 5S rRNA was determined using two independent methods and compared with other plant 5S rRNAs. It shows more than 90% sequence homology with gymnosperm 5S RNAs. The free energy (delta G) analysis of 5S rRNAs from gymnosperms, angiosperms and the other higher plants revealed that the free energy of this ribosomal RNA decreases with evolution.

Segmental Duplications in the Human Genome

The review considers the structure, evolution, and possible mechanisms of formation and spreading of intrachromosomal and interchromosomal segmental duplications (SD), which account for more than 5% of the human genome. Most SD consist of multiple modules, which occur in several copies in different genome regions. SD are preferentially located in pericentric and subtelomeric regions, which are least studied on the human chromosomes. Homologous recombination between SD results in various chromosome rearrangements, contributing to the genome instability and the origin of several human hereditary disorders.

Tandem and Interspersed Repeats Contribute to the Mosaic Structure of Segmental Duplications in the Human Genome

Intrachromosomal and interchromosomal segmental duplications account for more than 5% of the human genome. To analyze the processes resulting in the complex mosaic structure of duplicons, a draft human genome sequence was searched for duplicated segments of a genomic fragment of the pericentric region of the chromosome 21 short arm. The duplicons found consist of modules having paralogs in various genome regions. Module ends are flanked with various tandem or interspersed repeats, which are more unstable as compared with unique sequences. In most cases, the boundaries of duplicated segments exactly coincide with or are in close proximity to hot spots of various rearrangements within repeats or boundaries between repeats and unique sequences or between two different repeats. Homologous recombination between repetitive elements was assumed to be the major mechanism contributing to the mosaic structure of duplicons.

Directed cleavage of ribopolynucleotides with nucleases restricted by multiple modification of substrate

Simultaneous exhaustive modification of cytidine and uridine residues of rRNA with methoxyamine and sodium metabisulfite renders adjacent phosphodiester bonds resistant to pancreatic and T2 ribonucleases. Another method of T2 RNAase restriction is modification of cytidine with methoxyaminebisulfite followed by modification of guanosine residues with beta-ethoxy-alpha-ketobutyraldehyde. Mild alkaline treatment leads to demodification of uridine and guanosine residues leaving intact modified cytidine residues, thus providing a means of stepwise, directed cleavage of the polynucleotide. The series of combined cleavage procedures and methods of isolation of oligo(C), oligo(G) and oligopyrimidine tracts, as well as the procedure of selective cleavage at uridine residues elaborated in the course of the present studies may serve as a basis for more rational procedures of RNA sequencing.