Evolutionary conservation of repetitive sequence expression in sea urchin egg RNA's.

Cloned repetitive DNA sequences were used to determine the number of homologous RNA transcripts in the eggs of two sea urchin species, Strongylocentrotus purpuratus and S. franciscanus. The eggs of these species contain different amounts of RNA, and their genomes contain different numbers of copies of the cloned repeats. The specific pattern of repetitive sequence representation in the two egg RNA's is nonetheless quantitatively similar. The evolutionary conservation of this pattern suggests the functional importance of repeat sequence expression.

Message sequences and short repetitive sequences are interspersed in sea urchin egg poly(A)+ RNAs.

At least half of the mass and most of the different single-copy maternal mRNA sequences in the egg are covalently associated with transcripts of short repetitive sequences. Only a restricted group of the diverse genomic repeat families are significantly represented. The messages fall into several hundred sets, each containing transcripts from a different repeat family.

Repetitive sequence transcripts in the mature sea urchin oocyte.

The expression of interspersed repetitive sequences in the RNA of mature sea urchin oocytes was investigated. 3H-DNA tracers representing short interspersed repetitive sequences a few hundred nucleotides long, and long repetitive sequences approximately 2000 nucleotides long, were prepared from genomic DNA of the sea urchin, Strongylocentrotus purpuratus. These tracers were reacted with excess RNA from the mature oocyte. About 80% of the reactable short repeat tracer and 35% of the long repeat tracer hybridized. Thus most of the repetitive sequence families in the short repeat tracer are represented in oocyte RNA, and transcripts complementary to both strands of many repeat sequences are present. The kinetics of the reaction show that some transcripts are highly prevalent (greater than 10(5) copies per oocyte), while others are rare (approximately 10(3) copies per oocyte). Nine cloned repetitive sequences were labeled, strand-separated and reacted with the oocyte RNA. Transcripts of both strands of all nine repeats were found in the RNA. The prevalence of transcripts of the cloned repeat families varied from approximately 3000 to 100,000 copies per oocyte. Studies with both cloned and genomic tracers show that transcript prevalence is independent of the genomic reiteration frequency of the transcribed repetitive sequences. Most of the families represented by prevalent transcripts have fewer than 200 copies per haploid genome. The RNA molecules with which the cloned repeats react are at least 1000-2000 nucleotides in length. Other experiments show that a majority of the members of repeat families represented by prevalent transcripts in the oocyte RNA are interspersed among single-copy sequence elements in the genome.

A foreign beta-globin gene in transgenic mice: integration at abnormal chromosomal positions and expression in inappropriate tissues.

We have investigated the chromosomal location, inheritance, and expression of a cloned rabbit beta-globin gene introduced into the mouse germ line by microinjection into mouse eggs. Experiments utilizing in situ hybridization to metaphase chromosomes show that the gene has integrated into one or two different chromosomal loci in each of five mouse lines analyzed. Each locus contains between three and forty copies of the foreign DNA sequence arranged in a tandem array, and the sequences at each locus are stably inherited as a single Mendelian marker. Neither globin mRNA nor polypeptides encoded by the rabbit beta-globin gene are detected in erythroid cells in the seven transgenic lines examined, indicating that the expression of the foreign gene is not correctly regulated. However, in two of the mouse lines, rabbit beta-globin transcripts are found at a low level in specific, although inappropriate, tissues: skeletal muscle in one line and testis in another line. These unusual patterns of beta-globin gene transcription are heritable traits in the two mouse lines and may result from the beta-globin gene's integration at abnormal chromosomal positions.

Specific representation of cloned repetitive DNA sequences in sea urchin RNAs.

Nine cloned repetitive sequences were labeled, strands-separated and individually hybridized with RNA extracted from the nuclei of gastrula stage sea urchin embryos and of adult sea urchin intestine cells. The concentration of transcripts complementary to each cloned sequence was measured by RNA excess hybridization kinetics and by a DNA excess titration method. Transcripts of certain of the repeat families are present at over 100 times the concentration of transcripts of other families in each RNA. The set of repetitive sequence families highly represented in intestine nuclear RNA is different from that highly represented in gastrula nuclear RNA. Together with the results obtained with mature oocyte RNA and presented in the accompanying paper by Costantini et al. (1978), these findings show that quantitative patterns of repetitive sequence representation in RNA are specific to each cell type. Both strands of all of the nine cloned repeats are represented at some level in all the RNAs studied. Usually, though not always, the concentration of transcripts complementary to the two strands of each repeat do not differ by more than a factor of two. The cloned tracers do not react with polysomal messenger RNA, and the nuclear RNA molecules with which they hybridize are many times larger than the repetitive sequences themselves.

Transgenic mice expressing human sickle hemoglobin are partially resistant to rodent malaria.

The polymorphic frequency of the gene for beta s-globin involved in the generation of sickle trait and sickle cell anemia in the human population is caused by the enhanced resistance of sickle trait individuals to Plasmodium falciparum malaria, as supported by epidemiologic and in vitro studies. However, the mechanism for the protective effect of sickle hemoglobin in vivo has not been fully defined. The generation of transgenic mice expressing high levels of human beta s- and alpha-chains has allowed us to study this phenomenon in vivo in an experimental model. We infected the transgenic beta s mice with two species of rodent malaria and found a diminished and delayed increase in parasitemia as compared with controls. This is in contrast to our previous studies involving the introduction of a beta A transgene, which does not alter the infection. The use of this model allowed us to address the question of the mechanism of protection against malaria in mice expressing sickle hemoglobin. We find that splenectomy of transgenic mice completely reverses the protection against Plasmodium chabaudi adami infection. The results reported have shown a relationship between the presence of the beta s gene product and partial resistance to malaria in an experimental model in vivo and shows that the spleen plays an important role in this protection.