Frog Genomes Research Articles

A family of Tc1-like transposons from the genomes of fishes and frogs: evidence for horizontal transmission

Tc1-like transposons are very widely distributed within the genomes of animal species. They consist of an inverted repeat sequence flanking a transposase gene with homology to the mobile DNA element, Tc1 of the nematode Caenorhabditis elegans. These elements seem particularly to infest the genomes of fish and amphibian species where they can account for 1% of the total genome. However, all vertebrate Tc1-like elements isolated so far are non-functional in that they contain multiple frameshifts within their transposase coding regions. Here I describe a Tc1-like transposon ( PPTN) from the genome of a marine flatfish species ( Pleuronectes platessa) which bears conserved inverted repeats flanking an apparently intact transposase gene. Closely related, although degenerate, Tc1-like transposons were also isolated from the genomes of Atlantic salmon ( SSTN, Salmo salar) and frog ( RTTN, Rana temporaria). Consensual nucleic acid sequences were derived by comparing several individual isolates from each species and conceptual amino acid sequences were thence derived for their transposases. Phylogenetic analysis of these sequences with previously isolated Tc1-like transposases shows that the elements from plaice, salmon and frog comprise a new subfamily of Tc1-like transposons. Each member is distinct in that it is not found in the genomes of the other species tested. Plaice genomes contain about 300 copies of PPTN, salmon 1200 copies of SSTN and frog genomes about 500 copies of RTTN. The presence of these closely related elements in the genomes of fish and frog species, representing evolutionary lines, which diverged more than 400 million years ago, is not consistent with a vertical transmission model for their distributions.

Y3 is the most conserved small RNA component of Ro ribonucleoprotein complexes in vertebrate species

YRNAs are small cytoplasmic RNAs that are components of the Ro ribonucleoprotein complex. This complex, which also includes the 60-kDa Ro protein, is a human autoantigen which is conserved among vertebrates, and is of unknown function. Multiple sequences with YRNA homology, known as YRNA-like sequences, have been detected in rabbit, mouse, duck, iguana and frog genomes with human Y cDNA probes. As judged by Northern blots of total RNA, however, not all of these genomic YRNA-like sequences are expressed. Complementary DNA and putative gene sequences for iguana Y3 (iY3) and iguana Y4 (iY4) Ro RNAs have been determined and used, along with previously sequenced human and frog Ro YRNA sequences, to construct the most likely Y3 and Y4 RNA secondary structures. The data presented indicate that Y3 is the most conserved Ro RNA, not only by its more consistent presence in other species, but also at the levels of sequence divergence and secondary structure similarity. The differences observed between the secondary structure solutions for the Y3 and Y4 Ro RNAs are consistent with the possibility that these RNAs perform different cellular functions.

Sequence and developmental expression of mRNA coding for a gap junction protein in Xenopus.

Cloned complementary DNAs representing the complete coding sequence for an embryonic gap junction protein in the frog Xenopus laevis have been isolated and sequenced. The cDNAs hybridize with an RNA of 1.5 kb that is first detected in gastrulating embryos and accumulates throughout gastrulation and neurulation. By the tailbud stage, the highest abundance of the transcript is found in the region containing ventroposterior endoderm and the rudiment of the liver. In the adult, transcripts are present in the lungs, alimentary tract organs, and kidneys, but are not detected in the brain, heart, body wall and skeletal muscles, spleen, or ovary. The gene encoding this embryonic gap junction protein is present in only one or a few copies in the frog genome. In vitro translation of RNA synthesized from the cDNA template produces a 30-kD protein, as predicted by the coding sequence. This product has extensive sequence similarity to mammalian gap junction proteins in its putative transmembrane and extracellular domains, but has diverged substantially in two of its intracellular domains.

Replication, integration and expression of exogenous DNA injected into fertilized eggs of Xenopus laevis

We have analyzed the fate of circular and linear DNA molecules following microinjection into the cytoplasm of fertilized eggs of Xenopus laevis. Recombinant plasmids containing sea urchin histone genes (pSp 102), Drosophila ADH genes (SAC-1), and SV40 (SV2 CAT) replicate during the development of the injected frog embryo. In contrast, pBR322 either as monomers or multimers does not appear to replicate as efficiently. Generally, injected circular DNAs were not detectable by the gastrula stage of development, although there were several examples in which these molecules persisted until larval stages. In 90% of the cases, injected linear DNAs persisted as discrete molecules into early embryonic stages. A portion of the DNA sequences complementary to injected linear and circular molecules was detected comigrating with the high-molecular-weight cellular frog DNA (48 kb or larger) from mid-cleavage stages onward. Restriction enzyme analysis of DNA from injected embryos suggested some copies of the injected DNAs were integrated into the frog genome. This occurred in about 10%–30% of the cases of injected circular DNA and approximately 60%–70% of the cases of injected linear DNA. We were able to rescue circular plasmids from the injected blastulae by retransforming Escherichia coli. Restriction enzyme analysis of this DNA suggested that the majority of injected circular DNAs were not modified following replication in the frog embryo. The DNA of Xenopus embryos was highly methylated. On the other hand, injected DNA sequences were not methylated de novo even after many replication cycles in the frog embryo. Ribonucleic acid (RNA) transcripts from the injected DNAs were detectable by the late blastula stage of development.

Transmission of integrated sea urchin histone genes by nuclear transplantation in Xenopus laevis.

Sea urchin histone genes contained in a recombinant plasmid pSp102 were microinjected into the cytoplasm of fertilized eggs of Xenopus laevis. By the late blastula stage, plasmid DNA sequences were detected comigrating with the high molecular weight cellular DNA (greater than 48 kilobases). Analysis of the DNA from injected embryos digested with various restriction endonuclease demonstrated that the injected DNA was integrated into the frog genome. Clones of embryos containing the pSp102 DNA sequences were produced by means of nuclear transplantation. Individuals of the same clone contain the pSp102 sequences integrated into similar chromosomal locations. These sites vary between different clones.