Igor B. Dawid

The structural organization of ribosomal DNA in drosophila melanogaster

The genes coding for the large rRNAs (rDNA) were purified from Drosophila melanogaster embryos by two different methods. Purified rDNA was analyzed by gel electrophoresis after digestion with restriction endonucleases and by electron microscopy. Each repeating unit of rDNA consists of a gene region that codes for a transcript which includes the 18S and 28S rRNA sequences plus a nontrascribed spacer. Analysis of rRNA/rDNA hybrids in the electron microscope reveals that two types of repeating units exist in the rDNA from this fly. One of these is characterized by insertions of various lengths within the 28S gene. Insertions range in size from about 0.5 kilo base pairs (kb) to 6.0 kb and occur in distinct size classes which are multiples of 0.5 kb. Figure 10 summarizes the structure of the rDNA repeating unit. Repeats with insertions constitute about two thirds, and repeats without insertions about one third of all repeating units in the sample studied where 86% of the rDNA is derived from the X chromosome nucleolus organizer. Statistical analysis of pairs of of nearest-neighbor repeats demonstrates that repeats with and without insertions are interspersed in the X chromosome nucleolus organizer, and that scrambling is close to that expected for random arrangement. Insertions are the major source of length heterogeneity in this rDNA. A minor length heterogeneity also exists in thie nontranscribed spacer of Drosophila rDNA as demonstrated by heteroduplex mapping of restriction fragments in the electron microscope.

Deoxyribonucleic acid in amphibian eggs.

DNA was isolated from the ovulated, unfertilized eggs of two amphibian species, Rana pipiens and Xenopus laevis . Control experiments showed that the eggs were free of bacterial contamination. The DNA content of eggs is 300 to 500 times the diploid complement present in somatic cells. This material was characterized as double-stranded DNA of high molecular weight by tests for deoxyribose, ultraviolet spectrum, melting curve, buoyant density in CsCl, sedimentation in sucrose gradients, chromatography on methylated albumin columns and template activity in the RNA polymerase reaction. Egg and liver DNA of R. pipiens have the same density in CsCl, but egg DNA of X. laevis is slightly more dense than the somatic DNA of that species. In hybridization experiments with the agar method, it was shown in competition experiments and by using enzymically synthesized RNA that egg DNA is complementary to a small fraction of liver DNA of the same species. The complementary sequences were not found in DNA from erythrocytes, which is otherwise indistinguishable from the DNA of other somatic tissues. Egg DNA is therefore a highly specialized DNA with an as yet undetermined function.lt is tentatively suggested that the bulk of egg DNA is of mitochondrial origin. Somatic DNA of R. pipiens contains 47% guanine plus cytosine, including 2% 5-methyl-cytosine; X. laevis DNA contains 42% guanine plus cytosine, including 1·4% 5-methyl-cytosine. The DNA content of X. laevis embryos from fertilization to stage 45 is reported. Experiments on the incorporation of 32 P into DNA during early cleavage show de novo synthesis of DNA, between fertilization and the 32-cell stage in R. pipiens .

Composition and structure of chromosomal and amplified ribosomal DNA's of Xenopus laevis

Abstract Ribosomal DNA of Xenopus laevis is a homogenous DNA component of high buoyant density which contains genes for ribosomal RNA. This DNA comprises about 0.2% of the chromosomal DNA of somatic cells (chromosomal rDNA † ). In oocytes of X. laevis , rDNA is amplified about 1000-fold and the additional rDNA copies (amplified rDNA) are located extrachromosomally. Both chromosomal and amplified rDNAs have been isolated and several of their properties compared. The single unequivocal difference between these DNAs is in their content of 5-methyl deoxycytidylic acid. Chromosomal rDNA contains 4.5% of its residues as 5-MeC, whereas amplified rDNA contains no detectable 5-MeC. The 5-MeC residues are located on both strands of chromosomal rDNA, including the gene regions which are transcribed in vivo . The base composition of the rDNAs is otherwise very similar; both contain 67% deoxyguanylic deoxycytidylic acids (with 13% of the latter methylated in chromosomal rDNA). Both rDNAs exhibit a biphasic melting curve reflecting two regions of the DNA with markedly different base compositions. Differences in their buoyant density and thermal stability are most likely due to their different contents of 5-MeC. The two rDNAs were compared by their ability to hybridize with rRNA and complementary RNA, which had been transcribed from either rDNA by Escherichia coli RNA polymerase. The stability of such hybrids and the nucleotide composition of the cRNAs transcribed from either rDNA were indistinguishable. The two rDNAs are therefore considered to be identical except for their difference in 5-MeC content. A model of rDNA is presented which includes data from several laboratories. The rDNA consists of a repeating unit of about 9 × 10 6 daltons which recurs about 450 times at each nucleolar organizer region. A gene sequence which is transcribed as the 40 s precursor rRNA molecule alternates with a sequence of about equal length which is not transcribed into RNA (“spacer region”). The model presents our estimates for the lengths and base composition of each region and for their arrangement in rDNA.

The molecular basis for length heterogeneity in ribosomal DNA from Xenopus laevis

The restriction endonuclease EcoRI cleaves Xenopus laevis ribosomal DNA twice in each repeating unit to yield two classes of fragments. One class is homogeneous in length and contains only gene sequences; the other class is heterogeneous in length and contains all of the non-transcribed spacer and some of the gene regions (see model in the Introduction). Four spacer-containing fragments of different length have been cloned in Escherichia coli. They have been compared by optical melting and by homoduplex and heteroduplex mapping in the electron microscope. The results show that two regions within the nontranscribed spacer account for the length heterogeneity. The region which varies most in length is adjacent to the transcription unit at its 5′ end, while the other variable-length region is near but not adjacent to the 3′ end of the transcription unit. Each of these variable-length spacer regions consists of internally repetitious simple sequences (“subrepeats”), which are probably shorter than 50 base-pairs in length. The length heterogeneity of rDNA repeating units is due to more or less copies of these subrepeats. These two regions of variable length are separated by a constant-length region. Another constant-length DNA region separates the 3′ end of the transcription unit from one of the variable spacer sequences. There is no evidence for subrepeats within these latter two spacer regions. It is proposed that the two variable-length regions within the spacer participate in and perhaps enhance the correction mechanisms which permit parallel evolution of tandem genes.

Vitellogenin in Xenopus laevis is encoded in a small family of genes

Vitellogenin, the yolk protein precursor, is produced in X. laevis liver from a 6.3 kilobase (kb) mRNA. Sequences of this mRNA have been transcribed into cDNA and cloned in E. coli. Some properties of 21 of these cloned DNAs, ranging in size from 1 to 3.7 kb, have been reported by Wahli et al. (1978b). This paper reports restriction endonuclease mapping, cross hybridization, heteroduplex mapping in the electron microscope and heteroduplex melting experiments with these DNAs. We conclude that the cloned DNAs fall into two main groups of sequences which differ from each other in approximately 20% of their nucleotides. Each main group contains two subgroups which differ from each other by about 5% sequence divergence. By hybridizing cloned DNAs with restricted genomic DNA, we showed that sequences corresponding to all four sequence groups are present in a single animal. Furthermore, we have obtained tentative evidence for the presence of large intervening sequences in genomic vitellogenin DNA. Analysis of R loop molecules demonstrated that all four sequences are present in the vitellogenin mRNA population purified from individual animals. While some alternate explanations are not entirely excluded, we suggest that vitellogenin is encoded by a small family of related genes in Xenopus.

Expression of ribosomal DNA insertions in drosophila melanogaster

Approximately half of the ribosomal genes on the X chromosome of Drosophila melanogaster are interrupted by an insertion of type 1. Nuclear RNA from D. melanogaster embryos was transferred to DBM paper and hybridized with cloned type 1 insertion sequences. With a DNA fragment derived specifically from large insertions, transcripts were detected between 5 and 10 kb. These insertion transcripts represent less than one RNA molecule per nucleus, which is more than three orders of magnitude below the concentration of nascent rRNA chains, as determined by kinetics of hybridization. With a DNA fragment derived from the right end of large insertions which is also complementary to short insertions, more discrete RNA bands appeared with sizes between 1 and 8.5 kb, representing altogether about 13 RNA molecules per nucleus. Insertion transcripts large enough to be potential precursors to 28S rRNA represent less than one molecule per nucleus. It was shown by sandwich hybridization that at least some of the insertion transcripts are derived from rDNA. No significant difference was found between insertion transcripts in RNA extracted from ovaries, embryos, larvae, pupae or adult flies. Unless a mechanism other than splicing is involved, ribosomal genes with insertions cannot contribute significantly to the synthesis of 28S rRNA. A cytoplasmic RNA approximately 1 kb long, which is complementary to a short insertion and to ribosomal gene sequences flanking both sides of the insertion, was found. The abundance of this short unspliced RNA is about 50 molecules per embryo cell.

Biogenesis of mitochondria duringXenopus laevis development

The unfertilized egg ofXenopus laevis contains 10 μg of mitochondrial protein and a cytochrome oxidase activity of 0.03 μatom oxygen/min/egg. During embryogenesis these values remain constant for about 2 days to stage 38, then increase coordinately and double by the feeding tadpole stage (45). The unfertilized egg contains 3.8 ng mitochondrial DNA. Incorporation of precursors into mitochondrial DNA proceeds at a low rate before stage 30 and increases more than 4-fold around stage 32. Apparently linear accumulation leads to a doubling of the mitochondrial DNA content by stage 45. Mitochondrial RNA consists predominantly of the two RNA components of the mitochondrial ribosome (rRNA),2 and of 4 S RNA. Eggs contain 13.0 ng of the large and 6.9 ng of the small mitochondrial rRNA. Mitochondrial rRNA synthesis begins, or at least accelerates 8-fold, at gastrulation (stage 10). Accumulation then proceeds at a constant rate, and by stage 45 the content of both mitochondrial rRNAs per embryo has doubled. Mitochondrial rRNA synthesized during embryogenesis is relatively stable, having a half-life of 50 hr. Mitochondrial rRNA is synthesized normally in anucleolate mutant embryos, indicating that the synthesis of cytoplasmic and mitochondrial rRNA is not coupled obligatorily. Mitochondrial 4 S RNA synthesis is absent or very low throughout early embryogenesis and is therefore not coordinated with the synthesis of rRNA. We conclude that the mitochondria synthesized during oogenesis and present in the egg are used during embryogenesis and are thus a storage product of eggs. Since the synthesis of different mitochondrial components initiates at different, characteristic stages of development we conclude that these components are under separate metabolic control.

Secondary structure maps of ribosomal RNA and DNA: I. Processing of Xenopus laevis ribosomal RNA and structure of single-stranded ribosomal DNA

Abstract Precursor and mature ribosomal RNA molecules from Xenopus laevis were examined by electron microscopy. A reproducible arrangement of hairpin loops was observed in these molecules. Maps based on this secondary structure were used to determine the arrangement of sequences in precursor RNA molecules and to identify the position of mature rRNAs within the precursors. A processing scheme was derived in which the 40 S rRNA is cleaved to 38 S RNA, which then yields 34 S plus 18 S RNA. The 34 S RNA is processed to 30 S, and finally to 28 S rRNA. The pathway is analogous to that of L-cell rRNA but differs from HeLa rRNA in that no 20 S rRNA intermediate was found. X. laevis 40 S rRNA ( M r = 2.7 × 10 6 ) is much smaller than HeLa or L-cell 45 8 rRNA ( M r = 4.7 × 10 6 ), but the arrangement of mature rRNA sequences in all precursors is very similar. Experiments with ascites cell 3′-exonuclease show that the 28 S region is located at or close to the 5′-end of the 40 S rRNA. Secondary structure maps were obtained also for single-stranded molecules of ribosomal DNA. The region in the DNA coding for the 40 S rRNA could be identified by its regular structure, which closely resembles that of the RNA. Regions corresponding to the 40 S RNA gene alternate with non-transcribed spacer regions along strands of rDNA. The latter have a large amount of irregular secondary structure and vary in length between different repeating units. A detailed map of the rDNA repeating unit was derived from these experiments. Optical melting studies are presented, showing that rRNAs with a high (G + C) content exhibit significant hypochromicity in the formamide/urea-containing solution that was used for spreading.

X and Y chromosomal ribosomal DNA of Drosophila: comparison of spacers and insertions.

Abstract In Drosophila melanogaster, the genes coding for 18S and 28S ribosomal RNA (rDNA) are clustered at one locus each on the X and the Y chromosomes. We have compared the structure of rDNA at the two loci. The 18S and 28S rRNAs coded by the X and Y chromosomes are very similar and probably identical (Maden and Tartof, 1974). In D. melanogaster, many rDNA repeating units are interrupted in the 28S RNA sequence by a DNA region called the insertion. There are at least two sequence types of insertions. Type 1 insertions include the most abundant 5 kilobase (kb) class and homologous small (0.5 and 1 kb) insertions. Most insertions between 1.5 and 4 kb have no homology to the 5 kb class and are identified as type 2 insertions. In X rDNA, about 49% of all rDNA repeats have type 1 insertions, and another 16% have type 2 insertions. On the Y chromosome, only 16% of all rDNA repeats are interrupted, and most if not all insertions are of type 2. rDNA fragments derived from the X and Y chromosomes have been cloned in E. coli. The homology between the nontranscribed spacers in X and Y rDNA was studied with cloned fragments. Stable heteroduplexes were found which showed that these regions on the two chromosomes are very similar. The evolution of rDNA in D. melanogaster might involve genetic exchange between the X and Y chromosomal clusters with restrictions on the movement of type 1 insertions to the Y chromosome.

Ribosomal DNA in Drosophila melanogaster. I. Isolation and characterization of cloned fragments.

Abstract Fragments of rDNA‡ from Drosophila melanogaster produced by the restriction endonuclease Eco RI were cloned in the form of recombinant plasmids in Escheriehia coli . Maps were prepared showing the location of the coding regions and of several restriction endonuclease sites. Most rDNA repeats have a single Eco RI site in the 18 S gene region. Thus, 19 of 24 recombinant clones contained a full repeat of rDNA. Ten repeats with continuous 28 S genes and repeats containing insertions in the 28 S gene of 0.5, 1 and 5 kb were isolated. The 0.5 and 1 kb insertion sequences are homologous to segments of the 5 kb insertions; because of this homology they are grouped together and identified as type 1 insertions. Four recombinant clones contain an rDNA fragment that corresponds to only a portion of a repeating unit. In these fragments the 28 S gene is interrupted by a sequence which had been cleaved by Eco RI. The interrupting sequences in these clones are not homologous to any portion of type 1 insertions and are therefore classified as type 2. In one of the above clones the 28 S gene is interrupted at an unusual position; such a structure is rare or absent in genomic rDNA from the fly. Another unusual rDNA fragment was isolated as a recombinant molecule. In this fragment the entire 18 S gene and portions of the spacer regions surrounding it are missing from one repeat. A molecule with the same structure has been found in uncloned genomic rDNA by electron microscopic examination of RNA/DNA hybrids.