Nucleic Acids Research: VOLUME 40 ISSUE 19 2012

Restriction iipt were aade by Southern blot analysis of tha Amy (a-a»yla»t) region in 7 strains of Q. welanogaster using endonucleasaa Sail, Xhol and EcoRI. These were compared to tha •ap of AD»65 which contains the cloned Amy region. Strains used produce either two amylase variants, a single variant, or no amylase, yet all 7 strains carry two Any gone* as inverted repeats at the Any locus. This and the orientation of the repeats reaeablea the situation in XDm6S. Moat restriction sites mapped are conserved but two strains contain a large insertion which differs in size and position between strains. A complex anomaly, probably an inversion, exists at the Amy locus in a null strain. Haps for our Amv' strain and the XDm6S clone are identical, the DMA of each having been derived from a Canton-S wild stock. Restriction and genetic maps of the Amy region were aligned and alleles aasigned to the proximal and distal genes, Amv-p and Amy~d. IHTRODUCTION In Drosophiia aelanoqaater. the structural gene locus for alpha-aaylase (Amy. 2-77.7) is in section 54A1-B1 <1> of the right arm of the second chromosome. Eight electrophoretlc variants of tha anylase protein have been described, and additional variation has been revealed on the basis of heat lability studies (reviewed in 2). Many strains homozygous for chromosome 2 •"" press two amylaaa ioozymes. This, together with a variaty of other genetic and biochemical evidence, has been interpreted as indicative of a duplication of the Amy structural gene, with each gene copy represented by a number of allozymic forms (3-5). Whether or not the Amy gene is duplicated in strains characterized by a single amylase isozyme has been, until recently, a moot point. The molecular cloning and characterization of sequences from the Amy locus of D. melanoqaster (1, 6) provided direct O IRL Pren Limited, Oxford, England. 5337 Nucleic Acids Research evidence that individuals fro* one atrain which product! two el«ctrophor«tic amylaae variants have two copies of the emylaaa structural gone at this locus. Flies of this atrain, Canton-3, produce amylasa ltozyiu numbered 1 and 3 which are products of the respective genes, Amy and Amy . The cloned Amy genes, in recomblnant phage ADa6S, encode the appropriate amylase laozyaea and are oriented in opposite directiona, separated by approximately 5 to 6 kb of DMA <6). The present study was designed to answer several important questions about the nature of the Amy locus. First, la it typical for strains of D. »olanoqaster to contain duplicated structural genes at the Any locus, even when characterized by a single aaylase lsozyae? Second, in straina that do contain the duplication, are the Amy genes oriented in the same directions aa those observed for Canton-S and ia the diatance separating the two copies the same? Third, in the Amy "null" strain of Ha]-Ahmed and Hlckey <7> ia there a structural rearrangement at the Amy locus which might be responsible for the lack of amylaae expression? Fourth, what degree of variability la associated with various Amy strains in terms of specific restriction endonuclease sltas within and flanking the Amy genes? Finally, what la the relative orientation of the Any locus with raapect to the genetic and cytologlcal maps of chromosome 2£, i••w which Amy gene is centromere proximal to the other? To Investigate tha above questions, we examined the physical organization of tha Amy locus in seven "isogenic" strains of Q. melanogaster by means of Southern blot analysis. MATERIALS AND HETHODS Fly Stocks. Six of the seven "isogenic" Amy strains of D. melanogaster utilized in this study (Table 1) were originally homozygous for chromosonea 1, £ and 3 » > g ) a n d have been maintained in this laboratory for many years by single pair matlnga. Tha exception to this is the "null" strain, which was derived from the Amy "null" strain of Haj-Ahmed and Hlckey (7) . This strain waa made