Ronald H. Reeder

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.

Ribosomal RNA synthesis in isolated nuclei

Abstract Isolated nuclei from Xenopus laevis tissue culture cells synthesize RNA when incubated in vitro. A major fraction of this RNA is ribosomal RNA as shown by its specific hybridization to purified ribosomal DNA. The synthesis of ribosomal RNA is completely insensitive to the inhibitor α-amanitin. This suggests that ribosomal RNA is synthesized by form I, the major α-amanitin-resistant RNA polymerase found in these nuclei, but cannot rule out the possible involvement of form III, a minor α-amanitin-resistant RNA polymerase which is also present. The ribosomal RNA made in isolated nuclei is transcribed only from the H-strand of ribosomal DNA (the same as in the intact cell) and primarily from the 18 s and 28 s gene region. Little, if any, spacer sequences are transcribed.

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.

Variants of the TATA-binding protein can distinguish subsets of RNA polymerase I, II, and III promoters

Transcription extracts prepared from yeast that are deficient in the TATA-binding protein (TBP or TFIID) are also impaired in specific promoter recognition by all three nuclear RNA polymerases (pol I, II, and III). Specific initiation can be rescued by the addition of purified recombinant TBP, demonstrating that pol I, II, and III all require this factor. A mutation of TBP has been identified that will function with pol I but not with pol II or III. Conversely, another mutation, which inactivates TATA element binding in vitro, will function with pol I and III promoters but is inactive for a pol II promoter. Thus, it is possible to identify TBP variants that will only function on different subsets of all nuclear promoters.

rRNA synthesis in the nucleolus.

The past year has seen advances in our understanding of three broad areas that concern ribosomal RNA production. It is becoming apparent that for a large number of eukaryotes, sequence elements that regulate ribosomal RNA transcription are arranged in a similar pattern. This conservation of arrangement implies conservation of regulatory mechanisms. Better understanding of the ribosomal gene transcription factors has emerged, and one factor has been purified and cloned. In vitro systems for processing ribosomal RNA are beginning to be developed, allowing the first direct proof that a small nuclear ribonucleoprotein (U3) is involved in ribosomal RNA processing.

Spacer sequences regulate transcription of ribosomal gene plasmids injected into Xenopus embryos

We have injected plasmids containing a repeating unit (spacer plus gene) of Xenopus laevis ribosomal DNA into fertilized eggs. Transcription on these plasmids begins at the same time as transcription on the endogenous ribosomal genes (late blastula stage). Previous work defined the ribosomal gene promoter as the region from -140 to +6 around the site of transcription initiation (Reeder et al., 1982; Moss, 1982; Sollner-Webb et al., submitted). We show here that the level of transcription of the injected ribosomal genes is strongly affected by spacer sequences far upstream of the promoter. Deletion of spacer sequences over 1150 bp from the initiation site reduces the transcription signal from injected plasmids by a factor of 5-10. We propose that the upstream spacer sequences act to influence the frequency of promoter activation.

Transcription of chromatin by bacterial RNA polymerase.

Abstract The ability of Escherichia coli RNA polymerase to transcribe chromatin from mouse and frog (Xenopus laevis) liver was examined. In mouse liver chromatin, E. coli polymerase did not transcribe the mouse satellite sequences, although it did transcribe them in deproteinized DNA. In confirmation of previous reports, unlabeled mouse liver nuclear RNA competes more efficiently for the hybridization of chromatin transcripts to bulk DNA than it does with transcripts of deproteinized DNA. However, when chromatin and DNA transcripts were hybridized to mouse DNA from which the satellite sequences had been removed, they hybridized and competed identically. It is concluded that this type of hybridization-competition experiment is a very insensitive way to assay the fidelity of chromatin transcription. In chromatin from the frog, both the genes for rRNA and the genes for 5 S RNA are transcribed but in an aberrant manner. Transcription occurred on both strands and in the gene as well as spacer regions. In the F1 hybrid between X. laevis andX. mulleri, transcription of theX. mulleri rDNA † is repressed by over 95% in vivo (Honjo & Reeder, 1973). In chromatin from such hybrid frogs, E. coli RNA polymerase transcribed at least 25% X. mulleri rRNA, indicating that the bacterial polymerase could not recognize the repression mechanism. These studies suggest that transcription of chromatin by E. coli RNA polymerase is much less accurate than previously claimed.

Preferential transcription of Xenopus laevis ribosomal RNA in interspecies hybrids between Xenopus laevis and Xenopus mulleri.

Abstract Ribosomal RNA synthesized by hybrid frogs from the cross between Xenopus laevis and Xenopus mulleri was analyzed by molecular hybridization with purified ribosomal DNA from each species. Although the 18 S and 28 S rRNA sequences are indistinguishable between these two species, the remaining 10% of the 40 S rRNA precursor molecule of each species hybridizes about tenfold more efficiently to homologous rDNA than to heterologous rDNA. Using an assay based upon this fact, we show that in hybrid frogs X. laevis rDNA is transcribed preferentially and X. mulleri rDNA is repressed. X. mulleri rDNA is repressed regardless of which species is the female parent. The repression is nearly complete throughout early embryogenesis until the swimming tadpole stage, after which a low level of X. mulleri rRNA synthesis is detectable in the total embryo population. Some adult frogs made no detectable X. mulleri rRNA, whereas others were found that synthesized substantial amounts. Transcription of X. mulleri rDNA is repressed in embryos from the cross of a X. laevis female, heterozygous for the rDNA deletion mutation, and a wild type X. mulleri male. Half of these embryos contain only X. mulleri rDNA. The X. mulleri rDNA is transcribed eventually in these embryos but the onset of rRNA synthesis is much later than in wild type X. mulleri embryos. In the reverse cross (female X. mulleri × male X. laevis heterozygote) turn-on of X. mulleri rRNA synthesis was not delayed. The results of these four types of crosses indicate that either X. laevis rDNA or X. laevis maternal cytoplasm can each repress expression of X. mulleri rDNA in hybrid embryos. In the presence of X. laevis rDNA the repression can be permanent. The repression by X. laevis cytoplasm is transient and usually reversible.

Regulation of RNA polymerase I transcription in yeast and vertebrates.

This article focuses on what is currently known about the regulation of transcription by RNA polymerase I (pol I) in eukaryotic organisms at opposite ends of the evolutionary spectrum--a yeast, Saccharomyces cerevisiae, and vertebrates, including mice, frogs, and man. Contemporary studies that have defined the DNA sequence elements are described, as well as the majority of the basal transcription factors essential for pol I transcription. Situations in which pol I transcription is known to be regulated are reviewed and possible regulatory mechanisms are critically discussed. Some aspects of basal pol I transcription machinery appear to have been conserved from fungi to vertebrates, but other aspects have evolved, perhaps to meet the needs of a metazoan organism. Different parts of the pol I transcription machinery are regulatory targets depending on different physiological stimuli. This suggests that multiple signaling pathways may also be involved. The involvement of ribosomal genes and their transcripts in events such as mitosis, cancer, and aging is discussed.

The arrangement of length heterogeneity in repeating units of amplified and chromosomal ribosomal DNA from Xenopus laevis

Non-transcribed spacer regions of Xenopus laevis ribosomal DNA have been found which vary in length between 1.8 × 106 and 5.5 × 106 daltons. Length variation of rDNA† repeats exists within a single nucleolar organizer. Amplified rDNA contains repeats of the same size classes but often in different abundance than the chromosomal rDNA of the same animal. If a certain repeat length is preferred during amplification in an individual, it is also preferred in siblings with the same chromosomal rDNA composition. Thus, preference for a size class in amplification is inherited. Some animals selectively amplify repeat lengths which are rarely found in their chromosomal rDNA; others amplify their most abundant size class. The intramolecular arrangement of length variability was analyzed by the electron microscopy of heteroduplex molecules. Long single strands from two separate preparations of amplified and chromosomal rDNA each were reannealed with an homogeneous cloned spacer-containing rDNA fragment (CD30), and the size of adjacent heteroduplex regions was determined. The arrangement of length heterogeneity is very different in the two types of rDNA. Most, if not all, tandem repeats along a single molecule of amplified rDNA are equal in length. This observation supports a rolling circle mechanism for amplification. In contrast, between 50% and 68% of adjacent repeats in a given molecule of chromosomal rDNA differ in length. For one of the chromosomal rDNA preparations analyzed, the frequency of non-identical nearest-neighbors is compatible with random scrambling of repeats of different lengths. This result bears on the mechanism by which tandem genes evolve. It rules out sudden correction mechanisms of tandem genes such as the “master-slave” or certain “expansion-contraction” models, which predict that tandem genes will be identical.