The nucleotide sequence of oocyte 5S DNA in xenopus laevis. I. The AT-rich spacer

The primary sequence of the principal spacer region in X. laevis oocyte 5S DNA has been determined. The spacer is AT-rich and comprises half or more of each repeating unit. The sequence is internally repetitious; most of it can be represented by the following set of oligonucleotides:

$\text{CAA}{}^{\mathrm{C}}{}_{\mathrm{A}}\text{GTTTTCAA}{}^{\mathrm{AA}}{}_{\mathrm{GG}}\text{TTTG}{}^{\mathrm{C}}{}_{\mathrm{A}}\text{A}{}^{\mathrm{G}}{}_{\mathrm{T}}\text{TTTT(T)}$

The spacer, which varies in length from about 360 to 570 or more nucleotides, can be subdivided into a region (A₂) which is variable in length in different repeating units, flanked by regions (A₁, A₃, B₁) which are relatively constant in length. The A₂ region consists, on the average, of 5–6 tandem copies of the oligonucleotide CAAAGTTT-GAGTTTT; variation in the redundancy of this oligonucleotide accounts for much of the repeat length variation in the genomic 5S DNA. Most copies of this oligonucleotide are identical, although several differing by 1 or 2 nucleotides have been detected in plasmid-cloned 5S DNA fragments. Regions A₁ and A₃ comprise a linear array of similar, but not identical, oligonucleotides; most repeating units contain very similar A₁ and A₃ sequences. Region B₁ is a sequence of 49 nucleotides immediately adjacent to the 5′ terminus of the 5S rRNA sequence. It is GC-rich, much less repetitive than the remainder of the spacer and contains several palindromes, but no regions of dyad symmetry. This sequence is identical in all six of the single cloned repeating units of 5S DNA analyzed.     

The cyclization of Xenopus laevis 5 S DNA

DNA from Xenopus laevis containing the sequences complementary to 5 S RNA has been studied by the formation of folded rings. Maximal cyclization for fragments 1 to 2 μm in length is 45 to 55%. Thus the efficiency of folded ring formation from this tandemly-repeating DNA is about 50%, assuming that all fragments are 5 S DNA. From the ring frequency as a function of the number of nucleotides removed from the 3′ terminals of the shear-broken fragments, one may calculate that the repeating sequence is approximately 750 nucleotides long, a number that agrees with earlier partial denaturation mapping. The circumference of the folded rings confirms this repeating length since most rings correspond to modular size classes of 0.25-μm increments. Fragments 12 μm long cyclize almost as readily as 1 to 2-μm fragments do. Therefore, the length of the regions (g-regions) containing the tandemly-repeating 5 S DNA is more than 12 μm. The folded rings are about as stable to linearization by increasing concentrations of formamide as the duplex DNA is to denaturation. This indicates that the local, non-transcribed, spacer portions which represent the majority (83%) of the nucleotides in the tandemly-repeating unit, are probably homogeneous in sequence. The exonuclease-treated 5 S DNA fragments cyclize more rapidly than phage T7 DNA, and the kinetics are in accord with theoretical expectation.     

The nucleotide sequence of the major beta-globin mRNA from Xenopus laevis.

The nucleotide sequence of a cloned fragment containing an almost complete copy of the mRNA encoding the major adult beta-globin polypeptide in Xenopus laevis, the South African Clawed Toad, is presented. A procedure for strand separation by hybridization to complementary mRNA was used to determine some of the sequence and this technique is described. The complete amino acid sequence of the polypeptide has been deduced and comparison with other vertebrate beta-globins reveals several highly conserved, and therefore potentially important, regions of the protein. The sequence of beta-globin mRNA has been determined in several mammals, and in the chicken. Thus we have searched for conserved regions in the non-coding portions of these mRNA sequences, which encode the same protein, but which have been evolving separately for several hundred million years.     

A pseudogene structure in 5S DNA of Xenopus laevis

The 5S DNA of Xenopus laevis, coding for oocyte-type 5S RNA, consists of many copies of a tandemly repeated unit of about 700 base pairs. Each unit contains a "pseudogene" in addition to the gene. The pseudogene has been partly sequenced and appears to be an almost perfect repeat of 101 residues of the gene. The order of components in the repeat unit is (5') long spacer--gene--linker--pseudogene (3') in the "+" strand (or H strand) of the DNA. The possible function of the pseudogene is discussed.     

The isolation and characterization of a second oocyte 5s DNA from Xenopus laevis

A second and minor DNA component containing 5s RNA genes has been purified from the genomic DNA of Xenopus laevis (XIt 5S DNA). Some of its physical and chemical characteristics are described. It contains a 5S RNA gene sequence which has some oocyte and some somatic-specific residues, as well as nucleotides which differ from both types of 5S RNA. There are about 2000 of these 5S RNA genes per haploid complement of DNA compared to about 24,000 of the principal oocyte 5S RNA genes. The multiple repeating units of XIt 5S DNA are homogeneous in length (about 350 base pairs). We present evidence that XIt 5S RNA is transcribed in ovaries but not in somatic cells; XIt 5S DNA may therefore be under the same control as the major oocyte 5S DNA.     

Genetic recombination of Xenopus laevis 5 S DNA in bacteria

The behavior in genetic recombination of Xenopus laevis 5 S DNA has been examined, with particular emphasis on the role of 15-base-pair tandem repeats in the A + T-rich spacer. Fragments of 5 S DNA were introduced into Escherichia coli cells as inserts in the recombination vectors, lambda rva and lambda rvb. Intermolecular recombinants were selected in which, because of properties of the phage vectors, the crossover event must have occurred within the 5 S DNA inserts. Inserts from individual recombinants have been characterized in detail. The effects of varying the number (n) of 15-base-pair repeats and the recombination capabilities of the phage and host have been investigated. In these crosses, unequal crossovers can occur, yielding inserts different in size from the parental inserts. When the number of 15-mers is large (n = 12 or 20), most of the unequal crossovers have occurred within the 15-mers, resulting in an altered n value, although other homologies within the 5 S DNA sequence can also support unequal events. Increasing n in the parental inserts modestly increases the overall frequency of recombination and the percentage of altered inserts. We conclude that, in a bacterial setting, the 15-base-pair repeats stimulate recombination only slightly by allowing alternative registers for heteroduplex formation. The degree of stimulation observed is less than predicted by one simple model.     

The nucleotide sequence of mannosyl-Q-containing tRNAAsp from Xenopus laevis oocytes

The nucleotide sequence of tRNAAsp from X. laevis oocytes was determined as being: (sequence in text) The tRNA is 75 nucleotides long. This sequence is very similar (75% to 97% identity) to all other eukaryotic tRNAAsp sequenced so far, except for the bovine liver tRNAAsp (32% identity). The relation between the presence of a mannosyl group on queuosine (Q) at position 34 and the nucleotide sequence of the anticodon loop is discussed.     

DNA methylation patterns in the 5S DNAs of Xenopus laevis

The frequency of cytosine methylation at specific sites in the somatic 5S DNA (X1s) and trace oocyte 5S DNA (X1t) of X. laevis has been determined using restriction enzymes that are inhibited by the presence of 5-methylcytosine (5mC) within their cleavage sequences. 5S DNA methylation patterns were determined in genomic DNA from mature red blood cells, which express neither type of 5S gene, and from liver, which expresses only X1s. All the sites examined in X1t are greater than 95% methylated in red cells and liver. In the X1s of red cells all the sites examined are methylated in greater than 95% of repeats, while in liver some sites are modified in only 90% of repeats. Repeats containing unmethylated sites are randomly distributed throughout the tandem arrays in both red cells and liver. The high levels of methylation for X1s are in marked contrast to the situation with other Xenopus genes which do have sites of significant undermethylation in tissues where they are active. Thus, undermethylation in active genetic regions may not be a general feature for all classes of eukaryotic genes.     

Nucleotide sequence encoding the 5'' end of Xenopus laevis 18S rRNA.

We have sequenced a region of cloned Xenopus laevis ribosomal DNA encompassing the last 24 nucleotides of the external transcribed spacer and the first 275 nucleotides of the 18S gene. The start of the 18S gene was identified by correlating the results obtained from RNA hybridization and fingerprinting with the DNA sequence. This 5' region of 18S rRNA contains five 2'-O-methyl groups and at least six pseudouridine residues. Several of these modified nucleotides are clustered into a relatively short region from nucleotides 99-124. Nucleotides 227-250 constitute a distinctive sequence of 24 consecutive G and C residues. Comparison with the first 160 nucleotides of a yeast 18S gene (25) reveals three blocks of high sequence homology separated by two short tracts where homology is low or absent. The external transcribed spacer sequences diverge widely from within a few nucleotides of the start of the 18S gene.     

A DNA binding protein from Xenopus laevis oocyte mitochondria

A DNA binding protein has been isolated, by affinity chromatography on DNA cellulose, from mitochondria and from purified mitDNA-protein complexes from oocytes of Xenopus laevis. This 12,500 daltons protein is polymeric in its native form and binds to DNA with a high efficiency. It exhibits an apparently preferential binding to the single-stranded fiber of the D loop structures.