Ruth Yokoyama

Rhodopsin from the fish, Astyanax: role of tyrosine 261 in the red shift.

PURPOSE To isolate and characterize the rhodopsin cDNA from the fish, Astyanax fasciatus, and to determine the effect of tyrosine 261 on its spectral tuning. METHODS The rhodopsin cDNA was cloned using reverse transcription-polymerase chain reaction amplification and then sequenced. A mutant, Y261F, was generated by site-directed mutagenesis. Both wild type and mutant were transiently expressed in COS-1 cells, regenerated with 11-cis retinal, and purified by immunoaffinity chromatography. Ultraviolet-visible spectrophotometry was used to determine wavelength of maximum absorption. RESULTS A fasciatus rhodopsin cDNA exhibits 80% amino acid identity with bovine rhodopsin. In contrast to all known rhodopsins, this rhodopsin contains a tyrosine instead of a phenylalanine at amino acid position 261. Indeed, this particular amino acid replacement has been implicated in the long wavelength absorption of the red cone pigment. Site-directed mutagenesis was used to change the Astyanax amino acid 261 to phenylalanine (Y261F). Expression of the Y261F mutant in COS-1 cells showed an absorbance maximum of 496 nm, compared to 504 nm for the wild type pigment. CONCLUSIONS A naturally occurring fish rhodopsin is red shifted about 8 nm due to one critical amino acid substitution.

Chlamydomonas Flagellar Outer Row Dynein Assembly Protein Oda7 Interacts with Both Outer Row and I1 Inner Row Dyneins

We previously found that a mutation at the ODA7 locus in Chlamydomonas prevents axonemal outer row dynein assembly by blocking association of heavy chains and intermediate chains in the cytoplasm. We have now cloned the ODA7 locus by walking in the Chlamydomonas genome from nearby molecular markers, confirmed the identity of the gene by rescuing the mutant phenotype with genomic clones, and identified the ODA7 gene product as a 58-kDa leucine-rich repeat protein unrelated to outer row dynein LC1. Oda7p is missing from oda7 mutant flagella but is present in flagella of other outer row or inner row dynein assembly mutants. However, Oda7 levels are greatly reduced in flagella that lack both outer row dynein and inner row I1 dynein. Biochemical fractionation and rebinding studies support a model in which Oda7 participates in a previously uncharacterized structural link between inner and outer row dyneins.

Isolation, DNA sequence and evolution of a color visual pigment gene of the blind cave fish Astyanax fasc1atus

Visual pigment genes have been isolated from a Mexican blind cave fish, Astyanax fasciatus. We report here the DNA sequence of one of these genes, which has 70% nucleotide similarity with both the human red and green pigment genes. This gene appears capable of encoding a functional protein and is probably responsible for the green-sensitive pigment found in the pineal organ of the blind cave fish (Tabata, 1982). The pattern of nucleotide substitutions most likely reflects functional adaptation of the visual pigment gene during the past 400 million years of fish evolution.

Elimination of DNA sequences during macronuclear differentiation in Tetrahymena thermophila, as detected by in situ hybridization

Previous studies have indicated that certain sequences in the micronuclear genome are absent from the somatic macronucleus of Tetrahymena (Yao and Gorovsky, 1974; Yao and Gall, 1979; Yao, submitted). The present study used in situ hybridization to follow the elimination process during the formation of the new macronucleus. Micronuclear-specific DNA cloned in recombinant plasmids was labelled with 3H and hybridized to cytological preparations of T. thermophila at various stages of conjugation. Despite a smaller size and lower DNA content, the micronucleus has more hybridization than the mature macronucleus. Hybridization initially increased in the anlage (newly developing macronucleus) to reach a maximal level right after the old macronuclei had disappeared. The hybridization in the anlage then decreased to a significant extent prior to the first cell division. The results suggest that the micronuclear-specific sequence is first replicated a few rounds before it is eliminated from the anlage, and the elimination process occurs without nuclear division.

Molecular characterization of a blue visual pigment gene in the fish Astyanax fasciatus

We report here the isolation and sequence determination of a gene closely linked to the Astyanax red visual pigment gene. Reverse transcription polymerase chain reaction assays show that this new gene (B23Aƒ ) and the previously characterized red and green visual pigment genes of Astyanax are all expressed in the eye. Phylogenetic analysis shows that B23Aƒ belongs to the group consisting of short wavelength‐sensitive pigment genes from different species and is most closely related to the goldfish blue visual pigment gene.

Multiple origins of the green-sensitive opsin genes in fish

Vertebrate opsins are divided into four major groups: RH1 (rhodopsins), RH2 (rhodopsinlike with various absorption sensitivities), SWS (short-wavelength sensitive), and LWS/MWS (long and middle-wavelength sensitive) groups. The green opsin genes (g101Afand g101Af) in a Mexican characin Astyanax fasciatus belong to the LWS/MWS group, whereas those in goldfish belong to the RH2 group (Yokoyama 1994, Mol Biol Evol 11:32–39). A newly isolated opsin gene (rh11Af) from A. fasciatus contains five exons and four introns, spanning 4.2 kilobases from start to stop codons. This gene is most closely related to the two green opsin genes of goldfish and belongs to the RH2 group. In the LWS/MWS group, gene duplication of the ancestral red and green opsin genes predates the speciation between A. fasciatus and goldfish, suggesting that goldfish also has an additional gene which is orthologous to g101Af and g103Af.

Molecular characterization of a class IV human alcohol dehydrogenase gene (ADH7)

Class IV alcohol dehydrogenase (ADH) is a form preferentially expressed in stomach. We report here the isolation and sequence determination of a novel human ADH gene (ADH7). Phylogenetic analysis strongly suggests that ADH7 is a functional class IV ADH gene.

The effect of social selection due to familial mental retardation on the marriage frequency of normal individuals

Abstract This paper compares the marriage frequencies of normal individuals with or without mental retardates in their nuclear families for the different combinations of sex and parental and sib phenotypes. Multivariate analysis indicates that there is a difference in the marriage frequencies of normal individuals with and without retarded family members caused by social response to the trait. This response differs according to the sex of the normal individual, although the social factors underlying this sex difference are not clear.