Transcription of repetitive sequences on Xenopus lampbrush chromosomes.

Abstract

We reinvestigated the lampbrush chromosomes of Xenopus laevis and found them well suited for the study of transcription by in situ hybridization to nascent RNA transcripts. Using this technique, we analyzed the transcription of three repetitive sequences that do not show any sequence homology and that differ in their degree of interspersion. We found that they are located on different parts of the chromosomes: two are clustered, one is interspersed. All three of these sequences are transcribed at the lampbrush chromosome stage and transcripts from both strands of each sequence can be detected. The amount of transcription is apparently not proportional to the number of copies of the repetitive sequence at a given chromosomal locus, suggesting that other sequences are involved in the regulation of their transcription. The large size of the newt chromosomes is correlated with a very large genome, which, however, is a distinct disadvantage for experiments involving cloning and characterization of specific DNA sequences. For this reason we felt that it would be useful to search for an experimental system with a considerably smaller genome but with cytologically usable lampbrush chromosomes. The obvious choice was Xenopus laevis. The Xenopus genome is under investigation in several laboratories and the lampbrush chromosomes have already been described (9, 10), although they have been generally considered poorly suited for detailed cytological studies. We reinvestigated these chromosomes and found them quite adequate for the study of transcription and protein distribution by in situ hybridization and indirect immunofluorescence, respectively. In this report, we demonstrate that some of the long transcription units on Xenopus lampbrush chromosomes contain repetitive sequences. Transcripts of repetitive sequences have been reported in a variety of tissues, including the lampbrush chromosomes of newt oocytes (11)(12)(13)(14)(15). Studies of amphibian lampbrush MATERIALS AND METHODSIn Situ Hybridization to Mitotic Chromosomes. Mitotic chromosome squashes were prepared from gut cells of adult Xenopus and hybridized in situ according to Pardue and Gall (16).In Situ Hybridization to Lampbrush Chromosomes. 3H-Labeled complementary RNA (cRNA) was prepared from singlestranded probes and was hybridized in situ to nascent RNA chains on the chromosomes according to Diaz et al. (12) and Pukkila (17).Preparation of Cloned DNA and Radioactive Probes. Xenopus DNA was cut with restriction endonuclease HindIII and fractionated on a malachite green gel (18). The fractions were then electrophoresed on an agarose gel. The 750-base-pair band that is visible in the first fractions of the gradient was eluted and cloned in plasmid pBR322 by standard procedures. The identity of the cloned DNA was confirmed by hybridization to genomic DNA in a Southern blot. The HindII1 fragment was then recloned in a phage M 13 vector. In the course of this work we learned that the same repetitive DNA fragment was independently cloned by Lam and Carr...

Transcription of highly repetitive tandemly organized DNA in amphibians and birds: A historical overview and modern concepts

“…For example, centromeric satellite repeats TkS1 in Triturus cristatus group and X1-741 in Xenopus laevis are transcribed on lampbrush chromosomes. 25,37 RrS1 repeat is also transcribed on lateral loops arising from centromeric regions of Pelophylax ridibundus and P. lessonae lampbrush chromosomes (Fig. 1B and C).…”

“…2). 1,25,[35][36][37][38][39] Pretreatment of the lampbrush chromosome preparations with RNase A, RNase T1 and DNase I confirmed probe hybridization to the nascent RNA transcripts. 1,35,37,39 It became evident that satellite DNA is expressed during the lampbrush stage regardless of where they localize on the chromosomes (Table S1).…”

“…1,35 Transcribed tandemly repetitive sequences X132A in X. laevis were found at the terminal regions of the chromosomes. 25 Crucially, transcripts of X132 repetitive family were originally found in the oocyte RNA and polyrRNA fractions of X. laevis by RNA dot blot hybridization. 43 RNA ISH results revealed transcripts of telomeric repeat at the termini of lampbrush chromosomes of anuran amphibians P. ridibundus and P. lessonae ( Fig.…”

“…36 One of the 2 satellite DNA sequences (sat G family), called pTvml, with presumably pericentromeric localization is transcribed on the lampbrush chromosomes of T. vulgaris meridionalis. 41 Repeats distributed throughout the genome, such as satellite 2 of the newt N. viridescens, 38 X132C in X. laevis 25 or satellite 1 in bullfrogs Rana catesbeiana and R. pipiens 39 are transcribed during the lampbrush stage. Transcription occurs on different types of repetitive DNA sequences dispersed throughout the P. waltl genome 42 and satellite DNA According to the "read-through hypothesis" transcription starts from upstream protein coding gene promoters and proceeds without interruption into adjacent non-protein-coding sequences including arrays of tandem repeats.…”

“…22,23 The amphibian genomes, as a rule, have high C-values and large fraction of tandemly repetitive sequences that facilitates investigation of satellite DNA transcription. 18,[24][25][26] Genomes of model organisms such as chicken (Gallus gallus) or clawed frog (Xenopus) are thoroughly characterized, enabling detailed studies of the tandem DNA repeats. 20,26,27 Birds and amphibians are of a special interest due to the widespread transcription of tandem repeats at the diplotene stage of oogenesis when chromosomes acquire specific lampbrush form.…”