Norihiro Okada

SINEs : Short interspersed repeated elements of the eukaryotic genome

Much of the eukaryotic genome is composed of a variety of repetitive sequences. Amongst these, there are two kinds of retroposons (sequence elements derived from nonviral cellular RNA): SINEs (short interspersed elements) and LINEs (long interspersed elements). Amplification of SINEs occurs in a single germ cell, and the members of SINEs spread and become fixed in populations through genetic drift. SINEs can be regarded as phylogenetic landmarks: they are specific to one species, a few species, a genus or in some cases a family, indicating a specific time of amplification during evolution. Recent studies concerning the structure and origin of many SINEs revealed that retroposons are more widespread in animal genomes than was previously thought.

Tetrahymena actin: Cloning and sequencing of the Tetrahymena actin gene and identification of its gene product

Actin is ubiquitous in eukaryotes, nevertheless its existence has not yet been clearly proven in Tetrahymena. Here we report the cloning and sequencing of an actin gene from the genomic library of Tetrahymena pyriformis using a Dictyostelium actin gene as a probe. The Tetrahymena actin gene has no intron. The predicted actin is composed of 375 amino acids like other actins and its molecular weight is estimated as 41,906. Both T. pyriformis and T. thermophila possess a single species of actin genes which differ in their restriction patterns. Northern hybridization analysis revealed that the actin gene was actively transcribed in vivo. To detect the gene product, we synthesized an N-terminal peptide of the deduced sequence and prepared its antibody. Using an immunoblotting technique, we identified Tetrahymena actin on a two-dimensional gel electrophoretic plate. The actin spot migrated near an added spot of rabbit skeletal muscle actin, but clearly differed from the latter in its isoelectric point and apparent molecular weight. The primary structure of Tetrahymena actin shares about 75% homology equally with those of other representative actins. This value is extremely low as a homology rate between known actins. Tetrahymena actin diverges not only in relatively variable regions of other actins, but also in relatively constant regions. The hydrophilicity levels of two regions (residues 190 to 200 and residues 225 to 235) are also quite different between the Tetrahymena actin and skeletal muscle actin. Thus, we conclude that actin is present in Tetrahymena, but it is one of the most unique actins among the actins known hereto.

Rodent type 2 Alu family, rat identifier sequence, rabbit C family, and bovine or goat 73-bp repeat may have evolved from tRNA genes

SummaryClose structural resemblances between several mammalian highly or moderately repetitive families and some specific tRNAs were detected. The rodent type 2 Alu family, rat identifier (ID) sequences, rabbit C family, and bovine or goat 73-bp repeat are most homologous with lysine tRNA5, phenylalanine tRNA, glycine tRNA, and glycine tRNA, respectively. The homologies extend to secondary structures, and the homologous nucleotides are located on nearly the same secondary structures. The repetitive families mentioned have a common structural organization, with a tRNA-like sequence devoid of an aminoacyl stem region. These features suggest that these repetitive families may be generated by nonhomologous recombination between a tRNA gene and a tRNA-unrelated block.

Close phylogenetic relationship between vestimentifera (tube worms) and annelida revealed by the amino acid sequence of elongation factor-lα

To clarify the phylogenetic position of Vestimentifera (tube worms), 346-bp fragments of the elongation factor-lα (EF-lα) gene (939–1286 according to the numbering of the human gene) of a vestimentiferan, Lamellibrachia sp., a sternaspid polychaete, Sternaspis scutata, an earthworm, Pheretima sp., and a gastropod, Alviniconcha hessleri, were sequenced. From the amino acid sequences of these EF-lα, and those of two other vertebrates and two arthropods, phylogenetic relationships were deduced by the maximum likelihood (ML) method, by which the phylogenetic tree can be inferred without assuming constancy of the molecular evolutionary rate. For the ML tree and all of seven alternative trees, whose log-likelihoods could not be discriminated from that of the ML tree by the criterion of the standard error, the vestimentiferan, the polychaete, and the oligochaete formed a clade, excluding the arthropods and the gastropod as outgroups. This result is convincing evidence that Vestimentifera are protostomes that are closely related to Annelida. The ML tree suggests that Vestimentifera are more closely related to Polychaeta than to Oligochaeta, though the data were not sufficient to discriminate these three groups at a significant level. From recent evidence such as morphological characteristics and molecular information, it may safely be said that vestimentiferans should be included in the Annelida provided this phylum contains polychaetes and oligochaetes.

Transfer RNA-like structure of the human Alu family: Implications of its generation mechanism and possible functions

SummaryStructural resemblance of the human Alu family with a subset of vertebrate tRNAs was detected. Of four tRNAs, tRNALys, tRNAIle, tRNAThr, and tRNATyr, which comprise a structurally related family, tRNALys is the most similar to the human Alu family. Of the 76 nucleotides in lysine tRNA (including the CCA tail), 47 are similar to the human Alu family (60% identity). The secondary structure of the human Alu family corresponding to the D-stem and anticodon stem regions of the tRNA appears to be very stable. The 7SL RNA, which is a progenitor of the human Alu family, is less similar to lysine tRNA (55% identity), and the secondary structure of the 7SL RNA folded like a tRNA is less stable than that of the human Alu family folded likewise. Insertion of the tetranucleotide GAGA, which is an important region of the second promoter for RNA polymerase III in the Alu sequence, occurred during the deletion and ligation process to generate the Alu sequence from the parental 7SL RNA. These results suggest that the human Alu family was generated from the 7SL RNA by deletion, insertion, and mutations, which thus modified the ancestral 7SL sequence so that it could form a structure more closely resembling lysine tRNA. The similarities of several short interspersed sequences to the lysine tRNA were also examined. TheGalago type 2 family, which was reported to be derived from a methionine initiator tRNA, was also found to be similar to the lysine tRNA. Thus lysine tRNA-like structures are widespread in genomes in the animal kingdom. The implications of these findings in relation to the mechanism of generation of the human Alu family and its possible functions are discussed.

A novel antibody wich precipitates 7.5S RNA is isolated from a patient with autoimmune disease

Abstract We have isolated a nobel antibody from a patient with autoimmune disease which reacts with the ribonucleoprotein complex containing 7.5S RNA. The 7.5S RNA consists of two species having slightly different electrophoretic mobilities. Fingerprinting analysis of these two species demonstrates that nucleotide sequences of the RNAs are very similar to each other. Nucleotide composition of the 7.5S RNA is found to be; A/U/G/C=20/18/32/30, indicating that the ratio of GC content of the RNA(62%) is relatively high. The RNA contains a pseudouridylic acid residue as a modified nucleotide. Immunofluorescence pattern stained with the antibody suggests that the ribonucleoprotein complex containing 7.5S RNA is located both in the nucleolus and the cytoplasm.

The 6S RNA transcribed from rodent total DNA in vitro is the transcript of the type 2 Alu family.

SummaryWe have performed in vitro transcription, using total DNA isolated from mouse and a cloned mouse DNA fragment containing a representative type 2 Alu sequence characterized by Kominami et al. (1983) as templates. From each template, 6S RNA was transcribed. The fingerprint of the 6S RNA produced from in vitro transcription of total DNA showed a very clear pattern of oligonucleotides. By comparing the oligonucleotides of the 6S RNA produced from each template, we demonstrated that the 6S RNA transcribed from mouse total DNA is in fact the transcript of type 2 Alu sequences. The composition of the oligonucleotides of the 6S RNA transcribed from mouse total DNA seems to reflect well that of the consensus sequence of the type 2 Alu family proposed by Krayev et al. (1982). In vitro transcripts of rat or hamster total DNA showed fingerprint patterns nearly identical to that of mouse DNA, confirming that rodents have equivalent type 2 Alu sequences whose transcripts have the same length and similar compositions of oligonucleotides. Thus total DNA transcription provides a novel approach to the detection and analysis of type 2 Alu sequences transcribed in vitro.

Pseudouridylic modification of a 6S RNA transcribed invitro from highly repetitive and transcribable (Hirt) sequences of salmon total DNA

Salmon total DNA was transcribed in a HeLa cell extract, and it was found that a distinct 6S RNA is transcribed by RNA polymerase III. By analogy with a 6S RNA transcribed from mouse total DNA in vitro, which we demonstrated to be the transcripts from type 2 Alu family (Sakamoto et al. (1984) Mol Gen Genet 194:1-6), the genes for the 6S RNA in the salmon genome are presumed to be reiterated in at least 10(4)-10(5) copies and to be dispersed among the genome. A fingerprint analysis showed that the nucleotide sequences of the genes for the 6S RNA are extensively conserved. Furthermore, we demonstrated that at least two positions in the 6S RNA are modified in vitro to pseudouridylic acid. The pseudouridylic acid residues are located in specific oligonucleotides and each of the modified nucleotides amounts to 0.5 to 1.0 mol per mol of RNA. The relationship between salmon highly repetitive and transcribable (Hirt) sequences and rodent type 2 Alu family, and the significance of the pseudouridylic modification in relation to the functional role of these sequences are discussed.

Evolution of Repetitive Sequences

Three families of tRNA-derived SINE (short interspersed element) in the genomes of salmonid species were isolated and characterized. These three families, the salmon Sma 1 family, charr Fok 1 family and salmonid Hpa 1 family, differ in sequence, but are all derived from a lysine tRNA or a tRNA species structurally related to lysine tRNA. These have been amplified at the specific stage of evolution of salmonid species. The average sequence divergence of the salmon Sma 1 family, charr Fok 1 family and salmonid Hpa 1 family are roughly 0.7%, 0.9%, and 3.1% respectively, showing that the Sma 1 family amplified the most recently and that the Hpa 1 family is the oldest. There is a parallel between these values and distribution at a different position in the phylogenic tree of these families in the salmonid species. Thus, the genome in the proto-Salmonidae was first shaped by amplification and dispersion of the salmonid Hpa 1 family, and then in the genome of some descendents the salmon Sma 1 or charr Fok 1 family was amplified and these genomes were again reshaped.