Lampbrush chromosomes. Asymmetric loops.

Although the study of lampbrush chromosomes has proved highly informative over the years, some aspects of their structure remain strangely enigmatic.

In situ hybridization of highly repetitive DNA to chromosomes of Triturus cristatus.

Highly repetitive DNA of C0t 0--0.2 was purified from whole DNA of Triturus cristatus carnifex, labelled by nick translation, and in situ hybridized to RNA transcripts on the loops of lampbrush chromosomes and to the DNA of mitotic chromosomes from intestinal epithelium from the same species. The labelled DNA bound to 20--30 loops on the long arms of lampbrush bivalent 1, a pair of loops near the centromere on bivalent 10, and a number of other loops most of which were localized in pericentric regions. In mitotic preparations the same labelled DNA bound to the heteromorphic regions of the long arms of both chromosomes 1, and to the centromeric regions of all chromosomes. Centromeric labelling was light on chromosomes 4 and particularly clear on the 3 shortest chromosomes of the set. The heavy labelling of the heteromorphic arms of chromosome 1 is discussed in relation to several other peculiar properties of these arms, including their extraordinary lengths, their Giemsa banding patterns, and the absence of meiotic crossing over. It is suggested that insofar as the results with DNA/DNA hybridization and mitotic chromosomes match those obtained with the DNA/RNA-transcript hybridization and lampbrush chromosomes, confidence in the latter technique may be increased accordingly.

Lampbrush chromosomes and gene utilization in meiotic prophase.

The main features of lampbrush chromosome organization are reviewed and the significance of RNA transcription on lampbrush loops is questioned. Special consideration is given to evidence for the transcription on lampbrush loops of satellite DNA, low copy number genes with defined functions, the histone genes, the 5S genes and the genes for ribosomal RNA. It is concluded that there is widespread but somewhat indiscriminate transcription on lampbrush loops of a range of repetitive DNA sequences, transcription of a wide range of mRNAs, continuous transcription of histone and 5S RNA, and low or aberrant transcription of ribosomal RNA. The 'read-through' hypothesis of lampbrush loop transcription is explained and evaluated, and some of the assumptions underlying it and the problems it raises are examined and discussed. The hypothesis requires that transcription starts at a normal promoter site for a functional gene situated at the thin end of a lampbrush loop, and that once started the transcribing polymerase cannot stop until it reaches another promoter that is already initiated or some condensed and untranscribable chromatin. The following questions are considered. Why should polymerase on a lampbrush loop disregard normal termination signals? Why should polymerase stop when it encounters another initiated promoter sequence? Why is the number of loops or transcription units related to genome size signifying, according to the read-through hypothesis, that oocytes from animals with large genomes have more active 'functional gene promoters' than those from animals with small genomes? Finally, some special situations where there is enhanced or reduced lampbrush activity are considered and their significance in relation to ideas about the function of lampbrush chromosomes is discussed. Specific examples include the frog, Ascaphus truei, whose oocytes have eight germinal vesicles each with a full complement of lampbrush chromosomes, another frog, Flectonotus pygmaeus, in which each oocyte starts with several thousand meiotic nuclei only some of which go into a lampbrush phase, and certain species of reptile in whose germinal vesicles the chromosomes never acquire a lampbrush form.

The biological significance of variation in satellite DNA and heterochromatin in newts of the genus Triturus: an evolutionary perspective.

The functional and evolutionary significance of highly repetitive, simple sequence (satellite) DNA is analysed by examining available information on the patterns of variation of heterochromatin and cloned satellites among newts (family Salamandridae), and particularly species of the European genus Triturus. This information is used to develop a model linking evolutionary changes in satellite DNAs and chromosome structure. In this model, satellites accumulate initially in large tandem blocks around centromeres of some or all of the chromosomes, mainly by repeated chromosomal exchanges in these regions. Centromeric blocks later become broken up and dispersed by small, random chromosome rearrangements in these regions. They are dispersed first to pericentric locations and then gradually more distally into the chromosome arms and telomeres. Dispersal of a particular satellite is accompanied by changes in sequence structure (for example, base substitutions, deletions, etc.) and a corresponding decrease in its detectability at either the molecular or cytological level. On the basis of this model, observed satellites in newt species may be classified as 'old', 'young', or of 'intermediate' phylogenetic age. The functions and effects of satellite DNA and heterochromatin at the cellular and organismal levels are also discussed. It is suggested that satellite DNA may have an impact on cell proliferation through the effect of late-replicating satellite-rich heterochromatin on the duration of S-phase of the cell cycle. It is argued that even small alterations in cell cycle time due to changes in heterochromatin amount may have magnified effects on organismal growth that may be of adaptive significance.

C value and cell volume: their significance in the evolution and development of amphibians.

Cell volume has been determined in 18 species of amphibian, ranging in C value from 1.4 pg to 62 pg DNA. There is a strong linear relationship between C value and both erythrocyte volume and erythrocyte nuclear volume. We have collected data on the timing of early embryogenesis from fertilization of the egg to the hatching tadpole in some amphibians ranging in C value from 1.4 pg to 83 pg. The species with large genomes take up to 24 times longer to reach a comparable state of development. Polyploid species develop faster than closely related diploid species. These data are discussed in relation to genome expansion and increase in cell cycle time as factors in the evolution of the Amphibia.

The development of lampbrush chromosome-type transcription in the early diplotene oocytes of Xenopus laevis: an electron-microscope analysis.

Oocytes of Xenopus laevis in pachytene and early diplotene of meiosis have been studied using the Miller spreading technique. Transcription first appears in germinal vesicles 25-40 micrometers in diameter, when the oocyte is in early diplotene. Transcription at this stage consists of arrays of short RNP transcripts, irregularly distributed along the DNP axis. Linear regression analysis has shown that many of these arrays are transcription units (Type I) with the transcripts having a common point of origin. The lengths of these early transcription units (mean = 7.06 +/- 5.06 micrometers), calculated from the linear regression data, are comparable to the lengths of transcription units from later stages, including Dumont stage 3. The polymerase granules of Type I transcription units are widely and irregularly spaced, having a mean spacing of 505 +/- 748 nm. More advanced transcription units (Type II, mean length = 8.72 +/- 3.77 micrometer) are usually found in the same chromosome set as the Type I units. Type II transcription units have closer and more regularly spaced polymerase granules than Type I transcription units (mean spacing = 92 +/- 49 nm). Both Type I and II transcription units have comparatively short RNP transcripts, the mean values for the slopes of their regression lines being 0.1336 and 0.1440 respectively. By the time the germinal vesicles are about 50-60 micrometers in diameter the transcription units have a quite different morphology (Type III). The lengths of the Type III transcription units are comparable to the Type I and II units, the mean length being 6.34 +/- 4.03 micrometers. The spacing of the polymerase granules in the Type III units is closer and more regular than the earlier stages (70 +/- 40 nm). Another significant difference between Type III and Types I and II transcription units is a decrease in the foreshortening of the Type III RNP transcripts. The mean slope of the regression lines for Type III transcription units is 0.2439. The morphological appearance of the Type III transcription unit is virtually identical to that of the transcription units from Dumont stage 3 oocytes, both with respect to the length and the spacing of the polymerase granules. However, the transcripts in Type III transcription units are still more foreshortened than those of Dumont stage 3 oocytes, having mean regression slopes of 0.4728. From the data obtained in the present study, it has been concluded that the pattern of lampbrush-type transcription is virtually fully established by the time most germinal vesicles are about 50 micrometers in diameter.

Chromomere number and its genetic significance in lampbrush chromosomes.

The chromosomes of three species of salamander belonging to the genus Plethodon have been studied with regard to the absolute length of the haploid set of lampbrush chromosomes and the number and distribution of chromomeres per laploid set of lampbrush chromosomes. Each aspect has been considered in relation to the substantial difference in genome size between P. cinereus (C = 20 pg), P. vehiculum (C = 36.8 pg) and P. dunni (C = 38.8 pg). --Karyotype analyses carried out on unfixed preparations of lampbrush chromosomes showed that the absolute length of a haploid complement of lampbrush chromosomes from medium-sized yolky oocytes is much greater for P. vehiculum and P. dunni than for P. cinereus. Nonetheless, the relative dimensions of chromosomes are nearly identical in all three species. --Chromomers were counted along the whole length of the 14th (shortest) bivalent, and the total number of chromomeres in the haploid set of lampbrush chromosomes was determined by extrapolation on the basis of the known relative length of the 14th bivalent in the respective species. Chromomeres were also counted in measured segments of undamaged lampbrush preprations in which all chromosomes could be clearly identified. The average distance between chromomeres (chromomere distribution) was determined and the total chromomere number was estimated on the basis of measured total length of lampbrush chromosomes in a preparation. Chromomere distribution is the same for lampbrush chromosomes from all 3 species, and since P. vehiculum and P. dunni have longer chromosomes than P. cinereus, it is clear that the former 2 species have many more chromomeres (60-70%) per haploid set of lampbrush chromosomes. The term chromomere was used here in the descriptive sense to define a discrete granule of deoxyribonucleoprotein on the axis of a lampbrush chromosome.--These findings are discussed in relation to what is known of the molecular organization of eukaryotic genomes, and in relation to ideas on gene action and transcription in lampbrush chromosomes.

Centromeric satellite DNA in the newt Triturus cristatus karelinii and related species: its distribution and transcription on lampbrush chromosomes.

Two abundant satellite DNA sequences have been identified in and cloned from the DNA of Triturus cristatus karelinii. The smaller of these with a repeat unit of 33 base pairs (bp) is designated TkS1, the larger with 68 bp is designated TkS2. These satellites are also present in DNA from T.c. cristatus, T.c. carnifex and T. marmoratus but in substantially lower copy number. In situ hybridisations to lampbrush chromosomes of T.c. karelinii and T.c. cristatus have shown that the satellites are concentrated in the heterochromatic centromere bars of T.c. karelinii and in a region around the centromere granule in T.c. cristatus. The satellites also bind specifically to the centromere regions of mitotic metaphase chromosomes. They do not bind to the heteromorphic arms of chromosome 1, which have previously been shown to be rich in highly repeated DNA. DNA/RNA-transcript in situ hybrids to lampbrush chromosomes with TkS1 suggest that this sequence is occasionally transcribed on lampbrush loops near the centromeres.

In situ hybridization of "nick-translated" 3H-ribosomal DNA to chromosomes from salamanders.

A technique is described for preparation of 3H-labelled DNA by nick-translation employing deoxyribonuclease I and DNA polymerase I. The labelled DNA can be obtained in high yield with specific activities of 10(6) cpm/mug or more. Ribosomal DNA, isolated from ovaries of young Xenopus laevis, and whole DNA from Plethodon cinereus were labelled in this way. The rDNA was used for in situ hybridization to meiotic chromosomes from P. cinereus, P. vehiculum and P. dunni. Autoradiographs of in situ hybrids were exposed for 5 to 10 days, by which time nucleolus organizer regions on the chromosomes of all 3 species were clearly and specifically labelled. In all cases, labelling was confined to a short region near the middle of the short arm of both halves of a medium length bivalent. It is concluded that nick-translation is a useful and altogether efficient method of labelling nucleic acids for subsequent use in experiments involving in situ hybridizations.

The arrangement and transcription of telomere DNA sequences at the ends of lampbrush chromosomes of birds.

The arrangement of loops and chromomeres at the ends of lampbrush chromosomes in four species of bird is described with reference to chromomeres, loops and transcription units. Unlike the situation described in lampbrush chromosomes of amphibians, the lampbrush chromosomes of birds end in a terminal chromosome with conspicuous loops emerging from it. The fine-scale morphology of the ribonuclear protein matrix of these terminal loops is different from that of the majority of loops elsewhere on the chromosomes. In many cases the loops associated with the terminal chromomere are open ended, emerging from the chromomere but not returning to it at the other end. The distal ends of terminal open-ended loops therefore represent the true ends of the chromatids that make up a lampbrush half-bivalent. The pattern of binding of three telomeric DNA sequence probes to the terminal regions of bird lampbrush chromosomes, under conditions of DNA/DNA and DNA/RNA transcript in situ hybridization has been investigated by fluorescence in situ hybridization. All three probes gave the same results. With DNA/DNA and DNA/RNA transcript hybridization, three classes of structure were labelled: the terminal chromomere, a small number of interstitial chromomeres and the terminal transcription unit on telomere loops. Labelling of telomere loops, but not of terminal or interstitial chromomeres, was eliminated by ribonuclease treatment before in situ hybridization. The labelled regions of telomere loops were spaced away from the labelled terminal chromomere by an unlabelled sub telomeric transcription unit. After DNA/DNA in situ hybridization, no labelled loops were seen. DNA/RNA transcript in situ hybridization with single-stranded hexamers of each strand of telomeric DNA showed that the terminal transcription unit on telomere loops represents transcription exclusively from the C-rich strand of the repeat outwards towards the end of the chromosome. It is concluded that transcription specifically of the C-rich strand of strictly terminal clusters of telomere repeats is an obligatory event on the lampbrush chromosomes of birds and is unlikely to represent indiscriminate readthrough from proximally located gene elements.

Satellite DNA is transcribed on lampbrush chromosomes.

During the lampbrush stage of oogenesis there is widespread transcription, and it has been estimated that the total amount of DNA transcribed may be an order of magnitude greater than that required to produce the necessary functional RNA for the oocyte. We therefore considered it likely that some of the transcribed sequences have little, if any, translational significance, and may include both middle repetitive and highly repeated, or satellite, sequences. Satellite DNA is generally defined as rapidly reannealing DNA which has a short basic sequence that is repeated millions of times in the genome, usually in tandem arrays. The short repeated length, coupled with the organisation of satellite sequences in high order molecular weight tandem arrays in heterochromatic regions, have been put forward as reasons for supposing that this type of DNA is not normally transcribed. We report here that we have looked for and found evidence of transcription of satellite DNA on lampbrush loops in oocytes of the crested newt, Triturus cristatus carnifex.

In situ hybridization of ribosomal DNA labelled with 125iodine to metaphase and lampbrush chromosomes from newts.

Methods are described for in situ hybridization of ribosomal DNA from Xenopus laevis, labelled in vitro with 125iodine, to mitotic and lampbrush chromosomes from Triturus cristatus carnifex. The hybridization reaction was carried out in a mixture containing 50% formamide, 4 X SSC, 0.1 M KI, at 37 degrees C, or in 2 X SSC, 0.1 M KI at 65 degrees C. Autoradiographs of mitotic metaphases from 2 males showed labelling over the middle of the short arm of one chromosome IX in each metaphase. In some cases, a region near the end of a longer chromosome was also labelled. In a lampbrush preparations, labelling was confined to a region identified as about 53 units, near the middle of the short arm of both halves of bivalent IX. The usefulness of the technique and the significance of the labelling of only 1 of the 2 chromosomes IX in mitotic preparations are discussed.

Interspecific "common" repetitive DNA sequences in salamanders of the genus Plethodon.

Intermediate repetitive sequences of Plethodon cinereus which comprised about 30% of the genomic DNA were isolated and iodinated with 125I. About 5% of the 125I-repetitive fraction hybridized with a large excess of DNA from P. dunni at Cot 20. About half of the 125I-DNA in the hybrids was resistant to extensive digestion with S-1 nuclease. The average molecular size of the S-1 nuclease-resistant fraction was about 100 nucleotide pairs. The melting temperature of the S-1 nuclease-resistant fraction was about 2 degrees lower than that of the corresponding fraction made with P. cinereus DNA. These results are taken to indicate the presence in the genomes of P. cinereus and P. dunni of evolutionarily stable "common" repetitive sequences. The average frequency of repetition of the common repetitive sequences is about 6,000 X in both species. The common repetitive fraction is also present in the genomes of other species of Plethodon, although the general populations of intermediate repetitive sequences are markedly different from one species to another. The cinereus--dunni common repetitive sequences could not be detected in plethodontids belonging to different tribes, nor in more distantly related amphibians. The profiles of binding of the common repetitive sequences to CsCl or CS2SO4-Ag+ density gradient fractions of P. dunni DNA suggested that these sequences consisted of heterogeneous components with respect to base compositions, and that they did not include large amounts of the genes for ribosomal RNA, 5S RNA, 4S RNA, or histone messenger RNA. In situ hybridization of the 3H-labelled intermediate repetitive sequences of P. cinereus to male meiotic chromosomes of the same species gave autoradiographs after an exposure of seven days showing all 14 chromosomes labelled. The pattern of labelling appeared not to be random, but was impossible to analyse on account of the irregular shapes and different degrees of stretching of diplotene and prometaphase chromosomes. In situ hybridization of the same sequences to meiotic chromosomes from P. dunni gave autoradiographs after 60 d exposure in which all chromosomes were labelled. These heterologous in situ hybrids can only have involved the "common" repetitive sequences.

Multiple ribosomal gene sites revealed by in situ hybridization of Xenopus rDNA to Triturus lampbrush chromosomes.

A variety of 3H-labelled ribosomal gene probes were hybridized in situ to the nascent transcripts of lampbrush chromosomes from the crested newt, Triturus cristatus carnifex. The probes were from Xenopus laevis and included rDNA isolated by CsCl gradient centrifugation, recombinant plasmids and purified restriction fragments of rDNA. All the probes gave essentially the same result. About 10-15 loop pairs were distinctly labelled in each preparation, almost all of them located on the heteromorphic arms (HTAs) of chromosome 1. Ribosomal gene probes were also hybridized in situ to the DNA of denatured mitotic chromosomes from some of the individuals used to provide lampbrush preparations. Minor, scattered sites of hybridization were found in the HTAs, but the main clusters of ribosomal genes were found on chromosomes 6 and/or 9, in agreement with previous determinations of nucleolus organizer position in this species. However, the nucleolus organizers were not sites of labelled loops in lampbrush transcript hybridizations.--We have incubated isolated lampbrush-stage nuclei in media containing alpha-amanitin and labelled RNA precursors. Although extrachromosomal nucleolar genes incorporated label, supposedly due to transcription by RNA polymerase I, no lampbrush loops were labelled.--It appears that in T. c. carnifex there are ribosomal gene sequences at the main nucleolus organizers and at a number of sites scattered along the HTAs. The ribosomal genes at the nucleolus organizers are not extended in the form of actively transcribing loops unlike the ribosomal sequences on the HTAs, which are heavily labelled in transcript hybridization. The ribosomal sequences on the HTAs appear not to be transcribed by the same RNA polymerase that transcribes the ribosomal genes of extrachromosomal nucleoli.

Ribosomal gene amplification in multinucleate oocytes of the egg brooding hylid frog Flectonotus pygmaeus.

The multinucleate oocytes of Flectonotus pygmaeus begin as cysts containing 2,000 or more meiotic nuclei. Each nucleus amplifies its ribosomal DNA early in oogenesis. The level of amplification is widely different from one nucleus to another, and ranges from less than 0.1 x C to more than 8 x C. The C value for this species is 1.7 x 10(-12) g of DNA. In oocytes of about 0.5 mm diameter the nuclei sort themselves out into an outer shell of several hundred nuclei that swell up to become much larger than the nuclei that remain in the inner compact mass. Later the outer nuclei continue to swell and decrease in number while the inner nuclei disintegrate and disappear. By the time the oocyte reaches. 1.2 mm diameter there are only a few large nuclei left and each has many large nucleoli and a full set of lampbrush chromosomes. Eventually, only one germinal vesicle nucleus is left, and this has all the characteristics of the single germinal vesicles that are typical of oocytes from other amphibians. It is suggested that the sorting out of nuclei into the inner mass and the outer shell of larger nuclei in middle sized oocytes is a consequence of the positions the nuclei happen to be in at the time, but that the "contest" for the role of germinal vesicle may be won by the nucleus of the outer shell that has the highest ribosomal DNA content.

Ordered arrangement and rearrangement of chromosomes during spermatogenesis in two species of planarians (Plathelminthes).

The pattern of distribution of telomeric DNA (TTAGGG), 28S rDNA, and 5S rDNA has been studied using fluorescence in situ hybridization (FISH) and primed in situ labelling during spermatogenesis and sperm formation in the filiform spermatozoa of two species of planarians, Dendrocoelum lacteum and Polycelis tenuis (Turbellaria, Plathelminthes). In both species, the positions of FISH signals found with each probe sequence are constant from cell to cell in the nuclei of mature sperm. Chromosome regions containing 5S and 28S rDNA genes are gathered in distinct bundles of spiral form. In early spermatids with roundish nuclei, the sites of a given sequence on different chromosomes remain separate. Centromeres (marked by 5S rDNA) gather into a single cluster in the central region of the slightly elongated sperm nucleus. During spermatid maturation, this cluster migrates to the distal pole of the nucleus. In Polycelis, telomeric sites gather into three distinct clusters at both ends and in the middle of the moderately elongated nucleus. These clusters retain their relative positions as the spermatid matures. All the chromosome ends bearing 28S rDNA gather only into the proximal cluster. Our data suggest that structures in the nucleus selectively recognise chromosome regions containing specific DNA sequences, which helps these regions to find their regular places in the mature sperm nucleus and causes clustering of the sites of these sequences located on different chromosomes. This hypothesis is supported by observations on elongated sperm of other animals in which a correlation exists between ordered arrangement of chromosomes in the mature sperm nucleus and clustering of sites of the same sequence from different chromosomes during spermiogenesis.

Characterisation of a short, highly repeated and centromerically localised DNA sequence in crested and marbled newts of the genus Triturus.

A 32-33 bp highly repeated DNA sequence, TkS1, has been isolated from genomic DNA of the newt Triturus karelini digested with the restriction endonucleases HaeIII or AluI. TkS1 is known to be localised in the centromeric heterochromatin of all the chromosomes in T. karelini and the related species T. cristatus. TkS1 has been shown to be present in varying amounts in the genomic DNA of a range of species of Triturus, including representatives of the two main subgenera Triturus and Palaeotriton. A programme of sequencing of monomers, dimers and trimers of TkS1 was carried out in order to determine the level of conservation of the sequence within and between species of Triturus. Altogether 204 monomer (32/33 bp) clones were made of TkS1 from three individuals of T. karelini, and one individual each of T. cristatus, T. carnifex, T. dobrogicus and T. marmoratus, all members of the subgenus Triturus and the cristatus species group. A number of dimer (64 bp) and trimer (96 bp) clones were also made from DNA of a single specimen of T. karelini digested with HaeIII or AluI. Three distinct types of TkS1 were identified in all species examined, except for T. marmoratus where only two of the types were found. The types were distinguished on the basis of certain recurring divergent patterns in monomers sequenced from T. karelini. Type 1 is mainly characterised by the presence of an AluI site at positions 24-27 and type 3 mainly by the presence of an additional base (C) at position 14. Type 2 normally lacks the AluI site and the C at position 14, as well as having a number of other distinguishing features. TkS1 and its three types have remained remarkably constant in sequence since before the divergence of T. marmoratus from other species in the cristatus species group, about 10 million years ago. Examination of all 204 monomer clones and comparison with consensus sequences for the three types shows less than 5% divergence at any one position in the sequence. There is good evidence from examination of dimer and trimer clones of TkS1 that the different types are intermingled with each other, and all three types are likely to be present on all chromosomes. Dimeric (64 bp) TkS1 clones constructed from AluI fragments of T. karelini DNA show evidence of a trimeric (96 bp) "supertype" with the pattern type 1-type 3-type 1 that is much more common than would be expected on a random basis.(ABSTRACT TRUNCATED AT 400 WORDS)

Cytology, embryology, and evolution of the developmental arrest syndrome in newts of the genus Triturus (Caudata: Salamandridae).

We have examined embryonic development in three species (T. carnifex, T. cristatus, and T. marmoratus) of European newts of the genus Triturus (subgenus Neotriton) in which developmental arrest occurs in embryos that are homomorphic for a chromosomal heteromorphism involving chromosome 1 (Horner and Macgregor: J. Herpetol., 19:261-270, 1985). Embryonic arrest occurred during tailbud stages in all three species, but at a slightly earlier stage in T. marmoratus. Two phenotypes were identified among the arrested embryos. One of these is indistinguishable in embryonic morphology from normal embryos at all stages up to the time of arrest, but the other is characterized by a protruding yolk plug, which persists from the late gastrula/early neurula stage to the tailbud arrest stage and apparently interferes with normal morphogenesis. Evidence is presented that the two arrested phenotypes, which occur in approximately equal numbers, represent embryos that carry the two alternative homomorphic chromosome pairs of chromosome 1 heteromorphism. We conclude that developmental arrest reflects a balanced lethal heterozygosity probably resulting from an unequal exchange of genic material between the homologues of chromosome 1 which occurred in a common ancestor of the Neotriton species.

Cytological evidence of transcription of highly repeated DNA sequences during the lampbrush stage in Triturus cristatus carnifex.

Highly repeated, or satellite, DNA fractions have been isolated from total Triturus cristatus carnifex DNA by renaturation kinetics, caesium salt centrifugation and restriction endonuclease digestion. We have shown by DNA/DNA in situ hybridisation and autoradiography that all of these probes bind to C-band positive regions on mitotic or lampbrush chromosomes of T.c. carnifex. Under conditions of DNA to RNA-transcript in situ hybridisation labelled satellite DNA binds to nascent RNA transcripts that are still associated with the DNA axes of many lampbrush loops. The majority of the loops that label heavily in these experiments are located on the long arms of chromosome I, a region previously shown to be rich in highly repeated DNA and to have many of the properties of heterochromatin. These satellite DNA probes also label many loops on a comparable chromosome region in T. marmoratus, a species closely related to T. cristatus. However, in DNA/RNA-transcript hybrids to other more distantly related species of Triturus, there are no chromosome regions that have the same concentration of labelled loop pairs as the long arms of T.c. carnifex and T. marmoratus, although some loop pairs do label. We have cloned two satellite sequences in pBR322, and have obtained the same results using these pure probes as we obtained using satellite probes isolated by other techniques. These results demonstrate unequivocally that satellite DNA is transcribed on lampbrush chromosomes during oogenesis in crested newts.

Transcription of lampbrush chromosomes of a centromerically localized highly repeated DNA in pigeon (Columba) relates to sequence arrangement.

A highly repetitive, centromerically localized DNA sequence (PR1) has been isolated from the genomic DNA of two species of pigeon (Columba livia and C. palumbus). PR1 is approximately 900 bp long. It includes a sequence that is similar to the CENP-B box of mammals. It represents about 5% of the genome in C. livia and 2% in C. palumbus. In both species, tandem arrays of PR1 form part of larger repeating units. The organization of PR1 repeats and the larger repeating units is strikingly different in the two species. The large repeating units in C. livia include long (at least 14 units) tandem arrays of PR1 interspersed with relatively short intervening sequences. The large repeats of C. palumbus have much shorter (4 units or fewer) PR1 arrays interspersed with longer sections of non-PR1 DNA. PR1 is transcribed on short lampbrush loops in the centromeric regions of all lampbrush bivalents of C. palumbus. In C. livia, it is not transcribed at any of the major pericentromeric sites at which it is known to be present, although it is transcribed at one minor centromeric site on chromosome 2. It is proposed that transcription of the noncoding PR1 sequence on lampbrush chromosomes of pigeons relates to its genomic organization. The proposal is discussed with regard to the 'read-through' hypothesis for transcription on lampbrush loops.