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Biomedical subjects

Igor V Makunin

Publications and source records attributed to Igor V Makunin.

7 recordsLinked to original sources

Non-coding RNA.

The term non-coding RNA (ncRNA) is commonly employed for RNA that does not encode a protein, but this does not mean that such RNAs do not contain information nor have function. Although it has been generally assumed that most genetic information is transacted by proteins, recent evidence suggests that the majority of the genomes of mammals and other complex organisms is in fact transcribed into ncRNAs, many of which are alternatively spliced and/or processed into smaller products. These ncRNAs include microRNAs and snoRNAs (many if not most of which remain to be identified), as well as likely other classes of yet-to-be-discovered small regulatory RNAs, and tens of thousands of longer transcripts (including complex patterns of interlacing and overlapping sense and antisense transcripts), most of whose functions are unknown. These RNAs (including those derived from introns) appear to comprise a hidden layer of internal signals that control various levels of gene expression in physiology and development, including chromatin architecture/epigenetic memory, transcription, RNA splicing, editing, translation and turnover. RNA regulatory networks may determine most of our complex characteristics, play a significant role in disease and constitute an unexplored world of genetic variation both within and between species.

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Transposon-free regions in mammalian genomes.

Despite the presence of over 3 million transposons separated on average by approximately 500 bp, the human and mouse genomes each contain almost 1000 transposon-free regions (TFRs) over 10 kb in length. The majority of human TFRs correlate with orthologous TFRs in the mouse, despite the fact that most transposons are lineage specific. Many human TFRs also overlap with orthologous TFRs in the marsupial opossum, indicating that these regions have remained refractory to transposon insertion for long evolutionary periods. Over 90% of the bases covered by TFRs are noncoding, much of which is not highly conserved. Most TFRs are not associated with unusual nucleotide composition, but are significantly associated with genes encoding developmental regulators, suggesting that they represent extended regions of regulatory information that are largely unable to tolerate insertions, a conclusion difficult to reconcile with current conceptions of gene regulation.

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Genomic analysis of Drosophila chromosome underreplication reveals a link between replication control and transcriptional territories.

In Drosophila polytene chromosomes, most late-replicating regions remain underreplicated. A loss-of-function mutant of the suppressor of underreplication [Su(UR)] gene suppresses underreplication (UR), whereas extra copies of this gene enhance the level and number of regions showing UR. By combining DNA microarray analysis with manipulation of the number of Su(UR) gene copies, we achieved genomic-scale molecular identification of 1,036 genes that are arranged in clusters located in 52 UR chromosomal regions. These regions overlap extensively (96%) but are not completely identical with late-replicating regions of mitotically dividing Kc cells in culture. Reanalysis of published gene expression profiles revealed that genomic regions defined by replication properties include clusters of coordinately expressed genes. Genomic regions that are UR in polytene chromosomes and late replicated in Kc cell chromosomes show a particularly common association with transcriptional territories that are expressed in testis/males but not ovary/females or embryos. An attractive hypothesis for future testing is that factors involved in replication control, such as SU(UR), may interact physically with those involved in epigenetic silencing of transcription territories.

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Small regulatory RNAs in mammals.

Mammalian cells harbor numerous small non-protein-coding RNAs, including small nucleolar RNAs (snoRNAs), microRNAs (miRNAs), short interfering RNAs (siRNAs) and small double-stranded RNAs, which regulate gene expression at many levels including chromatin architecture, RNA editing, RNA stability, translation, and quite possibly transcription and splicing. These RNAs are processed by multistep pathways from the introns and exons of longer primary transcripts, including protein-coding transcripts. Most show distinctive temporal- and tissue-specific expression patterns in different tissues, including embryonal stem cells and the brain, and some are imprinted. Small RNAs control a wide range of developmental and physiological pathways in animals, including hematopoietic differentiation, adipocyte differentiation and insulin secretion in mammals, and have been shown to be perturbed in cancer and other diseases. The extent of transcription of non-coding sequences and the abundance of small RNAs suggests the existence of an extensive regulatory network on the basis of RNA signaling which may underpin the development and much of the phenotypic variation in mammals and other complex organisms and which may have different genetic signatures from sequences encoding proteins.

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Overexpression of the SuUR gene induces reversible modifications at pericentric, telomeric and intercalary heterochromatin of Drosophila melanogaster polytene chromosomes.

The SuUR (suppressor of underreplication) gene controls late replication and underreplication of DNA in Drosophila melanogaster polytene chromosomes: its mutation suppresses DNA underreplication whereas additional doses of the normal allele strongly enhances underreplication. The SuUR protein is localized in late replicating and underreplicating regions. The N-terminal part of the SuUR protein shares modest similarity with the ATPase/helicase domain of SWI2/SNF2 chromatin remodeling factors, suggesting a role in modification of chromatin structure. Here we describe novel structural modifications of polytene chromosomes (swellings) and show that SuUR controls chromatin organization in polytene chromosomes. The swellings develop as the result of SuUR ectopic expression in the transgene system Sgs3-GAL4; UAS-SuUR(+). They are reminiscent of chromosome puffs and appear in approximately 190 regions of intercalary, pericentric and telomeric heterochromatin; some of them attain tremendous size. The swellings are temperature sensitive: they are maximal at 29 degrees C and are barely visible at 18 degrees C. Shifting from 29 degrees C to 18 degrees C results in the complete recovery of the normal structure of chromosomes. The swellings are transcriptionally inactive, since they do not incorporate [(3)H]uridine. The SuUR protein is not visualized in regions of maximally developed swellings. Regular ecdysone-inducible puffs are not induced in cells where these swellings are apparent.

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Intercalary heterochromatin in Drosophila melanogaster polytene chromosomes and the problem of genetic silencing.

The morphological characteristics of intercalary heterochromatin (IH) are compared with those of other types of silenced chromatin in the Drosophila melanogaster genome: pericentric heterochromatin (PH) and regions subject to position effect variegation (PEV). We conclude that IH regions in polytene chromosomes are binding sites of silencing complexes such as PcG complexes and of SuUR protein. Binding of these proteins results in the appearance of condensed chromatin and late replication of DNA, which in turn may result in DNA underreplication. IH and PH as well as regions subject to PEV have in common the condensed chromatin appearance, the localization of specific proteins, late replication, underreplication in polytene chromosomes, and ectopic pairing.

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Microdissection and sequence analysis of pericentric heterochromatin from the Drosophila melanogastermutant Suppressor of Underreplication.

In the Suppressor of Underreplication( SuUR) mutant strain of Drosophila melanogaster, the heterochromatin of polytene chromosomes is not underreplicated and, as a consequence, a number of beta-heterochromatic regions acquire a banded structure. The chromocenter does not form in these polytene chromosomes, and heterochromatic regions, normally part of the chromocenter, become accessible to cytological analysis. We generated four genomic DNA libraries from specific heterochromatic regions by microdissection of polytene chromosomes. In situ hybridization of individual libraries onto SuUR polytene chromosomes shows that repetitive DNA sequences spread into the neighboring euchromatic regions. This observation allows the localization of eu-heterochromatin transition zones on polytene chromosomes. We find that genomic scaffolds from the eu-heterochromatin transition zones are enriched in repetitive DNA sequences homologous to those flanking the suppressor of forked gene [ su(f) repeat]. We isolated and sequenced about 300 clones from the heterochromatic DNA libraries obtained. Most of the clones contain repetitive DNA sequences; however, some of the clones have unique DNA sequences shared with parts of unmapped genomic scaffolds. Hybridization of these clones onto SuUR polytene chromosomes allowed us to assign the cytological localizations of the corresponding genomic scaffolds within heterochromatin. Our results demonstrate that the SuUR mutant renders possible the mapping of heterochromatic scaffolds on polytene chromosomes.

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