ABSTRACT
Nuclear domains can be divided into two major groups: those arising freely in nucleoplasm and those forming at specific chromosomal loci as a result of their activity. The advantages of giant transcriptionally active lampbrush chromosomes for the investigation of nuclear bodies formed in particular chromosomal regions have been demonstrated in a series of studies. We propose to use two strategies to analyze the loci of nuclear domains formation on lampbrush chromosomes typical for avian and amphibian oocytes. The first approach implies consecutive mapping of BAC-clones, containing the fragments of DNA assigned to genomic coordinates, in close proximity to the nuclear domains. The second approach is based on mechanical microdissection of chromosomal regions adjacent to a particular nuclear structure. DNA of dissected material can be amplified by PCR with degenerate primers and mapped by fluorescent in situ hybridization (FISH) on chromosomal spreads. Utilization of high-throughput sequencing (next generation sequencing, NGS) technologies also proves to be prospective for subsequent deciphering of regions underlying nuclear structures formation. Deciphered fragments can be aligned against reference genome assembly to define precisely the loci responsible for nuclear domains assembly. In this review, the possibilities of using two complementary strategies for investigation of nuclear domains associated with lampbrush chromosomes are demonstrated.
Subject(s)
Chickens/genetics , Chromosome Mapping/methods , Chromosomes/genetics , Genetic Loci , Models, Genetic , Animals , High-Throughput Nucleotide Sequencing/methods , In Situ Hybridization, Fluorescence/methodsABSTRACT
Avian oocyte chromosomes are transfomed into giant transcriptionally active lampbrush chromosomes (LBCs) at meiosis 1 diplotene. These chromosomes are a convenient tool for high-resulution cytogenetic analysis. Using differential staining with fluorochromes DAPI and CMA3, we have constructed detailed cytological maps for lampbrush macrochromosomes 1-5 and ZW of the Japanese quail Coturnix coturnix japonica. We also performed a comparative analysis ofmitotic chromosomes and LBCs corresponding to them. We estimated the decondensation coefficient during LBC formation and determined the centromere indices for mitotic and diplotene chromosomes and thus found that different chromosomes and chromosomal regions demonstrate unequal degrees of decondensation.
