Genetic Analysis O F Chromomere 3 D 4 in Drosophila Melanogaster . 11 . Regulatory Sites for T H E Dunce Gene '

Chromomere 3D4 of the X chromosome of D. melanogaster contains two genes, dunce (dnc ) and sperm amotile (sam). Mutations in dnc cause defects in memory formation and female fertility and reduce or eliminate the activity of a CAMP-specific phosphodiesterase designated form 11. A fine structure map of this region has been constructed showing the locations of two sam mutations, five dnc mutations and a newly identified locus designated cp t ro l of fertility (cf) that acts in cis to regulate the female sterility phenotype of dnc. The two sam mutations are separated by 0.02 f 0.01 cM, the rightmost being located 0.08 f 0.02 cM to the left of the null mutation dnc'". A cluster of null and form 11-defective dnc mutations is located 0.04 f 0.01 cM to the right of dnc'"". The cflocus is 0.06 f 0.02 cM to the right of this cluster. The location of the dnc and cf sites identify a region of approximately 0.10 cM that is required for proper expression of dnc+. The dncCK mutation, associated with a reciprocal translocation between 3L and the X, exhibits reduced form I1 activity and female sterility. This translocation breakpoint has been mapped to the left of the dnc+ gene and is near the breakpoint of Df(l)N64J15 which also reduces expression of dnc+. The effect of these independent chromosomal breaks on the dnc+ gene suggests the existence of a site to the left of dnc+ that is also required for proper expression of the gene. ROSOPHILA has two major forms of phosphodiesterase that differ in D molecular weight, substrate specificity, cation requirements and thermostability (KIGER and GOLANTY 1977, 1979; DAVIS and KIGER 1980). Form I, the cyclic nucleotide phosphodiesterase, hydrolyzes both CAMP and cGMP. This form of phosphodiesterase is activated by Ca2+ in the presence of calmodulin, a protein known to mediate the action of Ca2+ (KIGER and GOLANTY 1979; YAMANAKA and KELLY 1981; SOLTI et al. 1983; WALTER and KIGER 1984). Form 11, the CAMP-specific phosphodiesterase, is not activated by calmodulin or Ca2+ (SOLTI et al. 1983; WALTER and KIGER 1984), has a high requirement for Mg2+ and is more heat labile than form I (KIGER and GoKIGER and GOLANTY (1977) employed segmental aneuploidy in an attempt LANTY 1979). ' The previous paper in this series is SAL& DAVIS and KICER (1982). * Present address: Department of Biology, Princeton University, Princeton, New Jersey 08544. Genetics 108: 377-392 October, 1984. 378 H. K . SALZ AND JOHN A. KIGER, JR. to locate the structural genes for these phosphodiesterases and located that for form I1 in chromomere 3 D 4 . This region of the X chromosome is not required for viability, but it is essential for male fertility, normal female fertility and detectable form 11 phosphodiesterase activity (KIGER 1977; KIGER and GOLANTY 1979). Genetic analysis has revealed that chromomere 3D4 contains two genes: sperm amotile (sum), required for male fertility, and dunce (dnc), required for normal memory formation, female fertility and form 11 phosphodiesterase activity (BYERS, DAVIS and KIGER 1981; SALZ, DAVIS and KICER 1982). There are two lines of evidence suggesting that dnc is the structural gene for form I1 phosphodiesterase. First, there is a proportional relationship between the number of copies of chromomere 3D4 and form I1 activity (KIGER and GOLANTY 1979; SHOTWELL 1983). Second, two point mutations at the dnc locus, dnc' and dnc2, exhibit reduced levels of form 11 activity associated with changes in the physical properties of the enzyme. The dnc' mutant possesses a thermolabile enzyme, and the dnc' mutant's enzyme has abnormal kinetics (KAUVAR 1982; DAVIS and KIGER 1981). As both of these properties persist after extensive purification of form 11, it is probable, that these alterations are due to changes in the primary structure of the enzyme (KAUVAR 1982). The recombinational analysis presented here permits several dnc and sum mutations to be ordered in a fine structure map. During the course of this work the original dnc' strain was found to contain two closely linked mutations. Thermolability and reduction of form I1 enzyme activity appear to be associated with a mutation within a coding portion of the gene, whereas sterility is due to a mutation at a newly identified locus to the right of dnc, control of fertility (cf) . This locus acts in cis, suggesting that it is a regulatory site required for normal expression of the dnc+ gene. We propose that this site acts in conjunction with a functional dnc gene product to confer female fertility. The existence of two independent chromosomal breakpoints to the left of dnc that reduce the expression of the dnc gene suggests the possibility of additional regulatory sites to the left of the gene. MATERIALS AND METHODS Stocks: The deficiency and duplication chromosomes used have been described by KICER and GOLANTY (1977). The deficiencies used in this study are Df(I)N64i'6 (3C3-3D4), Df(I)N71h2'-5 (3C43D4) and Df(l)dm7"'g (3C12-3E4). The duplications used are Dp(I;2)w'5'b7(dnc+) (3C2-3D6), maintained in the second chrombsome balancer SMI;Cy D ~ ( I ; ~ ) W + ~ ' ~ ~ , and w+Y (2D1-3D1). The right breakpoint for the w+Y is that given by MCGINNIS, FARRELL and BECKENDORF (1980). The isolation and characterization of mutations in the dunce and the sperm-amotile genes have been described by SALZ, DAVIS and KICER (1982). The dnc mutations are balanced either with C(I)DX:y w f or FM7 (MERRIAM and DUFFY 1972). Evidence for an additional locus adjacent to dnc is presented in this report. This locus is designated cf (control of fertility). The mutant allde, cf ' , is present in the original dnc' chromosome; the other chromosomes carry what we call the wild-type allele and designate cf'. Whenever dnc' cf' and dnc' cf' are being compared, the stocks used are the parent stock, y w dnc' cf' f and y dnc' cf' ec f ) (no. 74 from the cross between y w dnc' cf' f and y dncM" cf+ ec f). The other X chromosomes used in this study are y dncM" ec J y w dncM14f; y dncM" ec, y w dncM" f; y w dncMLf'", y w dncCK f'". y w dnc2 v f ; dnc' ec f and dnc' cf' ec.