Oocyte-type 5S Research Articles

The reactivation of developmentally inert 5S genes in somatic nuclei injected into Xenopus oocytes.

In the frog Xenopus, the somatic-type 5S ribosomal RNA genes are active in all cells; the oocyte-type 5S genes are active in oocytes but not in somatic cells. A new method of native gel electrophoresis resolves the two types of 5S RNA which are of the same length but different sequence. When somatic nuclei were injected into oocytes, their inactive oocyte-type 5S genes often remained inactive, and had thus conserved their developmentally regulated condition. Other conditions, which include treatment with 0.35 M NaCl, reactivated the genes.

Nonrandom alignment of nucleosomes on 5S RNA genes of X. laevis

Mild digestion of Xenopus nuclei with micrococcal nuclease results in the cleavage of oocyte-type 5S RNA genes once every four nucleosomes, or about once per tandem repeating unit of 5S DNA. This specific cleavage pattern is observed with nuclei from somatic cells where oocyte-type 5S genes are never transcribed (blood and liver) and with cultured cell nuclei where these genes are in a DNAase I-sensitive chromatin conformation and low level transcription is observed. Cleavage of protein-free DNA with micrococcal nuclease does not result in a specific digestion pattern. The similarity of the nuclease-generated repeat length and the sequence repeat length of oocyte-type 5S genes suggested a sequence-specific arrangement of nucleosomes on these DNA sequences. Restriction endonuclease analysis indicates that micrococcal nuclease preferentially cleaves in a restricted region within the 5S repeating unit, about 200 bp from the single Hind III site. Using specific end-labeled DNA probes derived from cloned 5S DNA we can recognize at least four possible modes of organization of the nucleosomes on 5S DNA. In each of these phase arrangements, functionally significant regions of the 5S gene (start of transcription, middle control region and transcription termination site) are found in or near nucleosome linkers.

Recombination of a eukaryotic DNA in bacteria

Single, 824 bp repeating units of Xenopus laevis oocyte-type 5S DNA were inserted into the recombination vectors, λ rva and λ rvb. When the inserts had the same orientation with respect to the λ chromosomes, Spi - imm434 recombinants were recovered by selection on a P2, λ double lysogenic host. Because of the structure of the vectors, the crossover point in each recombinant must lie completely within the 5S DNA insert. The physical characteristics of these recombinants were determined by examination of restriction enzyme digests. By use of RecA mutant hosts and the Red - vector, λ rvc, recombination frequencies were measured separately for the bacterial and phage systems. Some of the recombination events resulted in 5S DNA inserts of altered length due to unequal crossovers within repeated sequences in the 5S DNA spacer. The occurrence of just such events in frog 5S DNA had been predicted, based on the structure of 5S DNA and evolutionary considerations.

Specific interaction of a purified transcription factor with an internal control region of 5S RNA genes

A factor necessary for the accurate transcription of cloned Xenopus 5S genes in vitro has been isolated from soluble extracts of X. laevis ovaries. The activity of the factor was monitored by its ability to facilitate transcription of exogenous 5S genes in unfertilized egg extracts which are otherwise incompetent for 5S gene transcription. The factor was purified via ion exchange chromatography, and apparently consists of a 37,000 dalton polypeptide. This factor is necessary for the transcription of both the oocyte-type and somatic-type 5S genes of Xenopus, but is not required for, and has no detectable effect upon, the transcription of a cloned Xenopus tRNA1Met gene. The site of action of the factor has been investigated using the "footprinting" method of Galas and Schmitz (1978). The factor binds specifically to intragenic regions extending, approximately, from nucleotide positions 45 to 96 on both somatic and oocyte-type 5S genes. Additionally, this binding occurs independently of, and is not altered by, the presence of purified RNA polymerase III or unfertilized egg extracts. The probable role of this factor in transcription initiation is discussed.

Biochemical research on oogenesis: Oocytes and liver cells of the teleost fish Tinca tinca contain different kinds of 5S RNA

Previtellogenic oocytes of Tinca tinca accumulate very large amounts of 5S RNA. We show here that 5S RNA stored in oocytes differs from liver 5S RNA in 3 out of 120 nucleotides. Liver and oocyte 5S RNAs, therefore, are produced by different genes. Both kinds of 5S genes are active in oocytes. However, only 5S RNA of the oocyte type accumulates in these cells. In Tinca tinca as in Xenopus laevis, oocyte-type and somatic-type 5S RNAs differ by three properties, ie., primary structure, conformation, and metabolic stability. Nucleotide substitutions occur in different positions in oocyte and somatic 5S RNAs of Tinca tinca and Xenopus laevis. We do not understand how different sets of nucleotide substitutions confer to 5S RNAs of both species similar properties in vivo, namely, increased metabolic stability.

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.

Repeating units of xenopus laevis oocyte-type 5S DNA are heterogeneous in length

The restriction enzymes Hind III and Hae III cleave Xenopus laevis 5S DNA at one and three sites, respectively, in each repeating unit of approximately 700 base pairs. The cleavage sites for both enzymes have been located within the repeating unit by denaturation mapping of the restriction fragments. The Hind III products and one of the Hae III fragments are variable in length, indicating heterogeneity in the length of the repeating unit in 5S DNA. This length heterogeneity is confined to the major A + T-rich spacer region. Repeating units differ from each other by discrete quanta of approximately 15 base pairs. The A + T-rich spacer has been shown to consist largely of tandem subrepeats of just this size ( Brownlee, Cartwright, and Brown, 1974). We suggest that the repeat-length heterogeneity is due to variable numbers of these subrepeats in the spacer regions of the major repeating units.