Heterochromatin regions of the chromosomes of the hen and Japanese quail in mitosis and at the lampbrush stage

The mitotic and lampbrush chromosomes of the domestic fowl and Japanese quail were analysed by fluorochrome staining technique. The lampbrush chromosomes of both the subjects displayed a typical "loop-chromomere" structure. Three distinct kinds of loops were distinguished in Gallus g. domesticus--normal, telomeric bows, and lumps. The former are distributed along the whole chromosome length. The latter and the bows were observed in subtelomeric and telomeric regions. By DNA/RNA specific acridine orange staining it was shown that each loop (especially, "lumpy" loops) contained a rich RNP matrix. A comparative analysis of the chromomycin A3/distamycin A banding pattern of mitotic and lampbrush chromosomes shows that the telomeric "bows" and "lumps" are special loops developed in telomeric heterochromatic bands. In Coturnix c. japonica, the CMA/DA-positive bands were not observed in telomeres of mitotic macrochromosomes, except a smallest band in the 2p-arm telomere. The absence of telomeric heterochromatic bands which can be visualized in the quail mitotic chromosomes coincides with the absence of "bow"-like loops. Only small lump-like structures were seen in some telomeres of macroautosomes. The biological significance of loop formation and RNA synthesis in heterochromatic band loops in growing oocytes is briefly discussed.     

Identification of the lampbrush chromosome loops which transcribe 5S ribosomal RNA in Notophthalmus (Triturus) viridescens

The loops which transcribe 5S ribosomal RNA in lampbrush chromosomes of the newt, Notophthalmus (Triturus) viridescens, were identified by hybridizing purified 5S DNA to nascent 5S RNA in situ. The genes which code for 5S RNA were found near the centromeres of chromosomes 1, 2, 6, and 7 by hybridizing iodinated 5S RNA to denatured lampbrush and mitotic chromosomes in situ. These genes and their intervening spacer DNA were isolated from Xenopus laevis using sequential silver-cesium sulfate equilibrium centrifugations. This purified 5S DNA was iodinated and hybridized to non-denatured lampbrush chromosomes in situ, where it bound to nascent 5S RNA on loops at the base of the centromeres of chromosomes 1, 2, 6, and 7. The number of 5S genes present in the haploid chromosome complement of N. viridescens was determined. — The 5S loops were chosen for study, since (1) the synthesis of 5S RNA has been demonstrated during the lampbrush stage, (2) both 5S RNA and 5S DNA could be isolated in pure form, and (3) the localization of the repetitive 5S genes could be verified by conventional in situ hybridization procedures. These methods may be applicable to the identification of other loops, leading to a better understanding of lampbrush chromosome function.     

Cytogenetic maps of lampbrush chromosomes of newts of the genus Pleurodeles: an algorithm of lampbrush chromosome identification in Pleurodeles waltl by immunocytochemical marker-loop staining with polyclonal anti-Ro52 antisera

Our work was aimed at developing a simple and effective method of identification of most or all chromosomes of Pleurodeles newts. To this end, we used DAPI staining of the chromomeres of newt lampbrush chromosomes and immunochemical reactions between the ribonucleoprotein (RNP) marker loops and polyclonal antibodies against human zinc-finger protein Ro52 (52-kDa Ro/SS-A). A method has been developed to obtain newt lampbrush chromosome preparations. Cytological maps of P. waltl chromosomes (Spanish population/subspecies) showing distributions of chromomeres and marker loops along the chromosome length were constructed.     

Molecular cloning of microdissected lampbrush loop DNA sequences of Drosophila hydei

We microdissected a Y chromosomal lampbrush loop pair from primary spermatocyte nuclei of Drosophila hydei and cloned the DNA directly at the microscale. Four of the 12 recombinant DNA clones recovered display in situ hybridization to mitotic metaphase Y chromosomes, preferentially in the chromosomal region identified as the origin of the lampbrush loop pair. All clones, however, also hybridize to autosomal and X chromosomal loci in polytene chromosomes. Y chromosomal DNA sequences of D. hydei again prove to be members of different families of repeated sequences distributed throughout the genome. These microcloning experiments, which were carried out under very unfavourable experimental conditions (low DNA content of the lampbrush loops in the presence of large amounts of RNA) prove that almost any chromosomal structure detected by light microscopy is directly accessible to molecular cloning experiments by micromethods.     

Molecular structure of the lampbrush loops nooses of the Y chromosome of Drosophila hydei

The molecular structure of the lampbrush loopforming fertility gene nooses from the short arm of the Y chromosome of Drosophila hydei is described on the basis of cloned DNA sequences which are characteristic for the sequence organization in the lampbrush loop. Y chromosomal lampbrush loops are organized into tandem repeat clusters of loop-specific repetitive DNA sequences and in interspersed repetitive DNA sequences with homologies elsewhere in the genome. In this paper, the basic properties of a repeat unit of the tandemly repeated sequence family ay1 are described. Moreover, it is shown that a loop contains several different domains carrying repeat clusters of the same repeated DNA family but with divergent sequence character. One of these clusters is characterized by an internal duplication of the basic repeat unit. We propose that the tandem repeat DNA family ay1 forms a frame of the lampbrush loop which is required for structural and functional reasons.