Subject: ms(3)k81 gene Dear Fly Base Curators, We wish to provide information for FlyBase on the identification of the ms(3)K81 gene which was reported in our previous publications. The gene was mapped through the characterization of deficiency alleles generated by excisions of P elements in 97D. These four alleles are ms(3)K812 through ms(3)K816 and are described in Yasuda, Schubiger, and Wakimoto, 1995. Genetics 140:219-229 . Existing mutations are strict paternal effect mutations. The mutant phenotype was fully rescued by transformation with a fragment containing nts# 22690254 through 22692698 (Release 3.1). This 2.4 kb fragment spanned from the 2nd exon of the Rb97D gene (CG6354) through the 2nd exon of the rough gene (CG6348). The transformation construct is a Bgl II fragment into the BamHI site of the pCaSpeR 4 vector. The orientation is nt 1-4793 of casper then 22690254-22693698 of the K81 region. The transformants were initially tested against the ms(3)K812 deficiency allele and have subsequently been tested as a trans-heterozygote against the other alleles and the larger Df(3R)roXB3. This rescue demonstrated that the ms(3)K81 gene is equivalent to CG14251 22691043-22691597. Characterization of a series of cDNA clones isolated from a testes-cDNA library revealed that there is no evidence of splicing of this message. Based on the cDNAs, the transcription start site is placed at \-42 and the poly-A tail is initiated 52nt downstream of the translation terminator sequence. With this placement, there are only 154bps between the Rb97D and K81 transcripts on the 5' end and only 260 bps between the K81 and rough transcripts on the 3' end. The only significant similarity to this gene in the data base is to a second gene in Drosophila, the CG6874 gene. <up>Reported at the 38th Annual Drosophila Research Conference, April 16-20, 1997</up>. The K81 transcript is testes-specific and is first observed in pupal stages. The transcript is only observed in the primary spermatocyte stages. Sequence analysis of two EMS-induced alleles reveals both result in nonsense mutations causing a shortened K81 product. The two ems alleles should be designated ms(3)K81Z3416 and ms(3)K81Z5795. The reference for the isolation of these alleles should be : Wakimoto, Lindsley, and Herrera (either ms in prep or personal communication). They both show the paternal-effect lethality characteristic of the other alleles. The Z3416 allele is predicted to produce a short 56aa polypeptide. The Z5795 allele is predicted to produce a K81 product that is missing only the last amino-acid. The C-terminal Glycine is found in the predicted CG6874 and in the K81 sequences from 5 other species. The sequence from a 6th species is highly conserved in the C-terminal region, but I do not yet have sequence information for the last 3 amino-acids. Please email us if you would like additional information. Thank you, Glenn Yasuda Chairman of Biology Assoc. Professor of Biology Biology Department, Seattle University 900 Broadway, Seattle WA 98122 Barbara Wakimoto Professor University of Washington Department of Biology Box 351800 Seattle, WA 98195-1800
