Phylogenetic relationships among taxa of the liverwort Conocephalum conicum (Conocephalaceae) revealed by psbA sequence.

The 5' flanking and coding regions of the psbA gene were sequenced to clarify relationships among several taxa discovered recently in thalloid liverwort Conocephalum conicum. Twenty six samples included in this study represent five "cryptic species" detected in worldwide collection, five groups discovered from Japan and finally three "chemo-types" with different dominant volatile component. Pairwise differences in nucleotide sequences of coding region between samples of Conocephalum conicum varied greatly depending on the combination, ranging from 0.001 to 0.018 per site. In total, seven amino acid substitutions were found among all samples. NJ and parsimony trees among the taxa were constructed for the first time. The phylogenetic relationships among taxa were basically consistent with those inferred from isozyme and morphological studies. Conocephalum conicum -species FS and T taxon found in Japan have identical sequence of psbA gene, suggesting that they are conspecific. Similarly, YFS and KYT taxa cluster together with FS cryptic species, and "chemo-types" I and II with "cryptic species" J. A chemotype III was very different from any other taxa of C. conicum. These results suggest that morphological species, Conocephalum conicum is highly differentiated at the molecular level and some of the taxa may in fact represents different species as previous studies suggested.

Geko, a novel gene involved in olfaction in Drosophila melanogaster.

To characterize genes involved in olfactory responses to chemical attractants, we screened 3000 P-element-tagged lines for their attraction to ethanol. Ten lines showed reduced levels of response, and revertants of these lines were obtained by excising the inserted P-element. The olfactory response of one line reverted to wild-type behavior compared to the original mutant line. The gene affected by this P-lacW insertion was named geko (gk). A 1.3-kb transcript was found to emanate from close to the P-insertion site, and the 5' upstream region was interrupted by the P-element. The amount of mRNA of gk gene in the P-lacW inserted line was about half that of the control strain. The response to ethanol of the gk(1) mutant was restored by transforming the genomic region containing this transcription unit. lacZ expression (stemming from this reporter-gene's presence in the transposon) was observed in the antenna and the antennal-maxillary complex (the olfactory organ of adults and larvae, respectively). gk mRNA was detected at the antenna and from other parts of the body. The deduced gk product showed no overall similarity to any reported amino-acid sequences.

Nucleotide variation and conservation at the dpp locus, a gene controlling early development in Drosophila.

A study of polymorphism and species divergence of the dpp gene of Drosophila has been made. Eighteen lines from a population of D. melanogaster were sequenced for 5200 bp of the Hin region of the gene, coding for the dpp polypeptide. A comparison was made with sequence from D. simulans. Ninety-six silent polymorphisms and three amino acid replacement polymorphisms were found. The overall silent polymorphism (0.0247) is low, but haplotype diversity (0.0066 for effectively silent sites and 0.0054 for all sites) is in the range found for enzyme loci. Amino acid variation is absent in the N-terminal signal peptide, the C-terminal TGF-beta peptide and in the N-terminal half of the pro-protein region. At the nucleotide level there is strong conservation in the middle half of the large-intron and in the 3' untranslated sequence of the last exon. The 3' untranslated conservation, which is perfect for 110 bp among all the divergent species, is unexplained. There is strong positive linkage disequilibrium among polymorphic sites, with stretches of apparent gene conversion among originally divergent sequences. The population apparently is a migration mixture of divergent clades.

Evolution of the LINE-like I element in the Drosophila melanogaster species subgroup.

LINE-like retrotransposons, the so-called I elements, control the system of I-R (inducer-reactive) hybrid dysgenesis in Drosophila melanogaster. I elements are present in many Drosophila species. It has been suggested that active, complete I elements, located at different sites on the chromosomes, invaded natural populations of D. melanogaster recently (1920-1970). But old strains lacking active I elements have only defective I elements located in the chromocenter. We have cloned I elements from D. melanogaster and the melanogaster subgroup. In D. melanogaster, the nucleotide sequences of chromocentral I elements differed from those on chromosome arms by as much as 7%. All the I elements of D. mauritiana and D. sechellia are more closely related to the chromosomal I elements of D. melanogaster than to the chromocentral I elements in any species. No sequence difference was observed in the surveyed region between two chromosomal I elements isolated from D. melanogaster and one from D. simulans. These findings strongly support the idea that the defective chromocentral I elements of D. melanogaster originated before the species diverged and the chromosomal I elements were eliminated. The chromosomal I elements reinvaded natural populations of D. melanogaster recently, and were possibly introduced from D. simulans by horizontal transmission.

The molecular analysis of brown eye color mutations isolated from geographically discrete populations of Drosophila melanogaster.

A large proportion of spontaneous mutations in Drosophila melanogaster strains of laboratory origin are associated with insertions of mobile DNA elements. As a first step toward determining whether spontaneous laboratory mutations are predictive for mutational events occurring in the wild, recessive brown (bw) eye color mutants were isolated. By inbreeding the progeny of wild-caught Drosophila melanogaster females, bw mutations were isolated from seven separate geographic sites distributed among Japan, California. Siberia and Hungary. Among a total of 14 mutations studied, no case of transposon mutagenesis was found. At least 4 mutations are associated with small deletions in the bw gene. The remainder are inseparable from wild-type bw by Southern analysis and are presumed to be basepair changes or very small indels. Although only two spontaneous bw mutants of laboratory origin have been analyzed molecularly, one is a mobile element insertion.

The relationship between DNA structural variation and activities of P elements in P and Q strains of Drosophila melanogaster.

To characterize the relationship between P element activities and their structures, we cloned P elements from genomic libraries of three isogenic P and Q strains derived from natural populations in Japan. These P elements were mapped with BamHI, AvaII and PstI and were classified by their size. The majority of P elements cloned were classified as either complete or relatively small P elements rather than medium size. The numbers of full length (2.9 kb) P elements per haploid genome of NP280 (P), AK194 (weak P) and WY113 (Q) were at least four, five and one, respectively. However, the 2.9 kb P element of WY113 was thought to be defective since this strain has no transposase activity. In our previous work, we demonstrated that the ORF 3-deleted P element is essential for P cytotype determination in WY113. A similar P element also exists in NP280, and this may have an important role for P cytotype determination in this strain. Two and one copies of the KP element, a deletion derivative of the P element, were found in NP280 and AK194, respectively. One of four complete P elements in NP280 was fully sequenced, and the base sequence was completely identical to that of p pi 25.1 originally derived from the U.S.A. This result is consistent with the notion that these P elements have a relatively recent origin in Drosophila melanogaster.

A novel eye morphology induced by a P element in somatic tissue of Drosophila melanogaster.

We found a specific eye morphology designated as Square, which is induced when some Drosophila melanogaster strains harboring P elements are crossed with the delta 2-3 strain carrying a modified P element, P[ry+, delta 2-3], which produces transposase in somatic tissue. This phenotype was dominant and also induced in the reciprocal crosses. Square was induced when the delta 2-3 strain was crossed with Q and M' strains such as the snw (M) strain carrying three small P elements but not with P strains. Inheritance of Square was also tested and its phenotype was not transmitted to the next generation. These results suggest that Square is caused by the transposition of P elements in somatic cells.

Excision of one of two defective P elements as the cause of alternate mutational events (sn+ and sne) of the singed bristle allele snw in Drosophila melanogaster.

The X-linked singed locus is concerned with the bristle phenotype and female sterility, and is known as a hot spot of P element insertion. A moderate allele of singed, singed-weak (snw) (Engels, 1979; 1984) is inserted with P elements. It is used as an index of P element activity, since it mutates at a high frequency to either a more extreme allele, singed-extreme (sne), or to a phenotype that is equivalent to the wild type (sn+) when an autonomous P element exists. We show here that snw is inserted with two defective P elements in reverse orientation, and the two alternate mutational events (sn+ and sne) are caused by the excision of one or the other of the P elements present in the singed gene. It is interesting that sn+ and sne are inserted with a single P element in the same position, but show very different phenotypes. The insertional sites of P elements in the singed locus possibly contain an unidentified repetitive sequence, which is repeated dozens of times per haploid genome of the wild-type strain Canton-S.

Repressor of P elements in Drosophila melanogaster: Cytotype determination by a defective P element carrying only open reading frames 0 through 2.

The P element is a type of transposable element in Drosophila melanogaster. Characteristics of the syndrome of "hybrid dysgenesis" are due to transposition of P elements, and the molecular mechanism for regulation of this transposition has been unknown. In this study a Q strain (which carries only defective P elements in its genome but still is able to repress the transposition of complete P elements although defective in transposase activity) was used to determine the structure of the P element with this repressor (or P cytotype-determining) domain. Examination of the cytotype and structure of the P elements of particular strains with reduced copy number of P elements showed that the P element with a repressor domain was defective, being deleted between bases 1991 and 2448. This region corresponds to most of the third intron [between open reading frame (ORF) 2 and ORF 3] as well as half the ORF 3 of an intact P element. Therefore ORF 3 was deemed to be unnecessary for repressor production.