Epistatic partners of neurogenic genes modulate Drosophila olfactory behavior.

The extent to which epistasis affects the genetic architecture of complex traits is difficult to quantify, and identifying variants in natural populations with epistatic interactions is challenging. Previous studies in Drosophila implicated extensive epistasis between variants in genes that affect neural connectivity and contribute to natural variation in olfactory response to benzaldehyde. In this study, we implemented a powerful screen to quantify the extent of epistasis as well as identify candidate interacting variants using 203 inbred wild-derived lines with sequenced genomes of the Drosophila melanogaster Genetic Reference Panel (DGRP). We crossed the DGRP lines to P[GT1]-element insertion mutants in Sema-5c and neuralized (neur), two neurodevelopmental loci which affect olfactory behavior, and to their coisogenic wild-type control. We observed significant variation in olfactory responses to benzaldehyde among F1 genotypes and for the DGRP line by mutant genotype interactions for both loci, showing extensive nonadditive genetic variation. We performed genome-wide association analyses to identify the candidate modifier loci. None of these polymorphisms were in or near the focal genes; therefore, epistasis is the cause of the nonadditive genetic variance. Candidate genes could be placed in interaction networks. Several candidate modifiers are associated with neural development. Analyses of mutants of candidate epistatic partners with neur (merry-go-round (mgr), prospero (pros), CG10098, Alhambra (Alh) and CG12535) and Sema-5c (CG42540 and bruchpilot (brp)) showed aberrant olfactory responses compared with coisogenic controls. Thus, integrating genome-wide analyses of natural variants with mutations at defined genomic locations in a common coisogenic background can unmask specific epistatic modifiers of behavioral phenotypes.

Alteration of 6-phosphofructo-1-kinase subunits during neonatal maturation of the rat cochlear cells.

During postnatal development of rat cochlear cells and the onset of hearing (10-23 days), the increasing endocochlear potential and energy requirements are largely provided by increased glucose utilization. It is well established that the ability of maturing rat tissues to use glucose is directly related to alteration of 6-phosphofructo-1-kinase (PFK) subunits. To gain insight into the alteration of PFK subunit levels in the cochlea from 6 to 60 days of age, PFK subunit types were measured in sections of paraffin-embedded temporal bone using IgG specific for each type of PFK subunit and quantified by computer image analysis. Although the L-type and C-type subunits did not exhibit statistically significant changes in the cochlear structures during maturation, the levels of M-type subunit in the stria vascularis cells, spiral ligament cell types I, II, and III, outer hair cells, inner hair cells, and support cells significantly increased. Also, the type IV and V spiral ligament fibrocytes during this period did not exhibit significant alterations of the M-type subunit. These data suggest that during neonatal development of the cochlear, the elevated levels of the M-type subunit are associated with increased glucose utilization and the onset of hearing.

Alteration of the levels of the M-type 6-phosphofructo-1-kinase mRNA isoforms during neonatal maturation of heart, brain and muscle.

During muscle, heart, and brain neonatal maturation, the capacity to utilize glucose in energy metabolism is directly related to the extent of accumulation of the 6-phosphofructo-1-kinase (PFK) M-type subunit. Neonatal development of other organs, such as liver and kidney, which are not characterized by large increases in the capacity to use glucose do not exhibit large increases in the M-type subunit protein. The presence of the M-type subunit in a PFK isozyme pool fosters a higher affinity utilization of carbohydrate and increased responsiveness to the levels of regulatory metabolites. To better appreciate this phenomenon, which is vital for normal development, the different isoforms of the M-type subunit mRNA's and alteration of their levels during maturation have been examined. Further, the potential promoter regions, i.e., the regions upstream from the sites of initiation of transcription, which are involved in expression of the different M-type subunit mRNA isoforms have been isolated, sequenced, and examined for possible transcription factor interaction sites. Using cDNA libraries produced from adult rat brain or skeletal muscle RNA, two primary forms of rat M-type subunit cDNA's were detected. Although the translated regions of these mRNA's were essentially identical, the 5'-untranslated region (5'-UTR) exhibited different lengths (90 or 59 bp) and sequences. Each M-type subunit cDNA had 10 common nucleotides immediately upstream from the initiator ATG, and the remaining 5'-UTR's had insignificant identity. A genomic fragment which interacted with probes complimentary to the sequences of the 5'-UTR of each M-type subunit mRNA isoform was isolated and sequenced by primer walking. It was discovered that the 5'-UTR of one of the mRNA's (proximal mRNA) was located immediately upstream from exon I and was apparently transcribed without splicing. Subsequently, the initial bp in the sequence of the other mRNA isoform (distal mRNA) was located 4010 bp upstream from the ATG in exon 1. Employing Reverse Transcription-Polymerase Chain Reaction using total RNA and scanning densitometry, the relative levels of the proximal and distal mRNA's during neonatal maturation of brain, heart, and muscle were measured. In these tissues, both forms of M-type subunit mRNA's were present, and during maturation tissue-specific differences were noted.

Membrane morphogenesis from cloned fragments of bacteriophage PM2 DNA that contain the sp6.6 gene.

The formation of new membrane vesicles normally occurs during eukaryotic organellogenesis and maturation of bacteriophage PM2. This virus was studied as a simple model for membrane morphogenesis. Previous biochemical and genetic studies suggest that a major structural protein of PM2, sp6.6, is an integral membrane protein involved in viral membrane morphogenesis. To establish the necessity of sp6.6 in membrane formation, restriction fragments of PM2 that contained the sp6.6 coding sequence were cloned into several plasmid vectors for expression in Escherichia coli. A construction in pBR322 containing two HindIII fragments of PM2 DNA caused production of intracellular membrane vesicles of the same size as those produced in the course of natural infection of Alteromonas espejiana. Similar results were obtained with a smaller construct of HindIII fragments in the plasmid vector pPL-lambda. Expression of sp6.6 was detected via incorporation of 35S-labeled methionine after SDS-polyacrylamide gel electrophoresis and with a specific rabbit antiserum on immunoblots. Other constructs did not produce recognizable vesicles or sp6.6. These results are the first to suggest that a hydrophobic membrane protein can cause development of new membrane structure.