In a sample of 79 genes with multiple introns, 33 showed significant heterogeneity in G+C content among introns of the same gene and significant positive correspondence between the intron and the third codon position G+C content within genes. These results are consistent with selection adding against preferred codons at the start of genes.

20-Hydroxyecdysone, but not juvenile hormone, regulation of yolk protein gene expression can be mapped to cis-acting DNA sequences. It is not clear whether regulation by 20-hydroxyecdysone is direct or indirect. Methoprene up-regulation is only observed when native yolk protein genes are assayed, suggesting that it may operate through influencing stability of the message.

Proteins which specifically bind the Yp1/Yp2 ovarian enhancer 1 element have been identified.

Site directed mutagenesis, protein binding and germline transformation experiments identify and characterise the activity of a simple mini-enhancer from the fat body enhancer (FBE region) consisting of a single binding site (dsxA) for the dsx protein and two others for other regulatory proteins (slbo and ref1). One copy of this enhancer is sufficient to direct the sex and fat body specificities of Yp1 transcription.

The o-r enhancer has four protein binding sites, three are involved in tissue specific transcription and the fourth is also necessary for sex specificity.

srp is expressed in the ovaries of adult flies where it produces an ovary specific protein isoform. The srp protein binds to a 12bp ovarian follicle cell-specific regulatory element located between the divergently transcribed Yp1 and Yp2. The 12bp element activates both in vivo and in vitro transcription of Yp1 and Yp2.

Insertion of the gypsy\su(Hw)BR into the Yp1 Yp2 intergenic region does not alter temporal or tissue-specific expression of the Adh or Ecol\lacZ reporter genes but does repress the Yp1 Yp2 fat body enhancer elements.

Analysis of transgenic lines containing altered Hsp70Bb promoters (fused to reporter gene Yp1), analysed for their ability to bind Hsf protein, indicates that at least three promoter sequences facilitate the binding of Hsf to chromatin, including binding sites for Trl, Tbp and RNA polymerase II.

The yolk protein genes Yp1 and Yp2 are only expressed in the ovary and fat body of females if they are supplied with proteinaceous food. Several regulatory regions have been shown to independently confer nutritional regulation on the expression of Yp1 and Yp2.

Deletion analysis of the Yp1 upstream sequences has demonstrated that additional sequences are functionally equivalent to the fat body enhancer (FBE) in Yp1 upstream region.

In vitro mutagenesis of dsx binding sites demonstrates that in males the dsx gene product acts to directly repress transcription of the yolk genes and in females the dsx gene product activates transcription by acting at the same sites in the fat body enhancer (FBE) driving expression of Ecol\lacZ. Through the male and female dsx proteins the sexual differentiation pathway is connected to a target gene by acting directly, but with opposite effects, on the gene.

Whereas in wild type ovarian follicles the Golgi apparatus and yolk spheres are preferentially labelled by osmium zinc iodide, in mutant follicles the Golgi apparatus and nearby vesicles showed dramatically reduced staining. Pattern of osmium zinc iodide staining in the cortical cytoplasm varies in relation to the number of yolk protein structural genes.

CrebA protein binds to the fat body specific enhancers of Dmul\Adh1, Adh, Yp1 and Yp2 and may be an important component of tissue specific regulation.

Newly synthesised yolk proteins in normal and mutant strains share secretory vesicles with putative vitelline membrane proteins. Translocation of follicle cell yolk protein is not through the membrane along the interfollicular spaces, but directly through plasmalemma facing the oocyte.

Ecol\lacZ reporter constructs carrying base substitutions in the AAE identify a negative regulatory element in the AAE to which the adult enhancer factor 1 (AEF1) binds. The AEF1 binding site, D.mulleri Adh-1 AAE and Yp1 gene fat body enhancer are related to a sequence recognised by the mammalian transcription factor C/EBP and a liver specific regulatory element of the human Adh gene. DNase I footprinting experiments reveal that AEF1 and C/EBP compete for adjacent binding sites in the fat body enhancers, AEF1 can displace bound C/EBP from its sites.

AEF1 binds specifically to fat body enhancer of Adh and Yp1.

Using Yp1 reporter gene constructs distal and proximal regions of the Hsp70 promoter have been identified that are required for formation of paused RNA polymerase II, the polymerase is transcriptionally engaged but paused about 25 nucleotides from the start site of Hsp70.

Evolutionary conservation of specificity of yolk protein uptake by the oocyte is studied throughout Diptera.

Female flies with varying numbers of Yp1, Yp2 and Yp3 genes have been generated. Each yolk protein gene makes an equivalent contribution to the fecundity and fertility of the female, and they do not individually provide unique functions to the embryo. The number of eggs laid by a female depends on the number of genes encoding yolk proteins present in the genome, and the probability of an egg producing an adult depends on the number of yolk protein genes present in the mother.

The male and female products of dsx when expressed in E.coli bind specifically to the fat body enhancer (FBE) of Yp1 and Yp2. This demonstrates a direct interaction between the sex determination hierarchy and a target gene.

The role of the ovary and nutritional signals in the regulation of Yp1 expression in the fat body has been studied.

Yp1 and Yp2 are transcribed in the same sub-populations of ovarian follicle cells. This expression is directed by two enhancers: ovarian enhancer 1, located in the 1226bp intergenic region, and ovarian enhancer 2 located within the first exon of Yp2.

Two cis-acting regions influence the transcription of both Yp1 and Yp2 in the ovaries. One is located in the 1224bp intergenic region and determines the stage and cell type specificity of ovarian transcription. The other is in the first intron of Yp2 and acts across the Yp2 promoter region to stimulate Yp1 transcription in ovaries.

A sequence-specific DNA-binding protein (Irbp) that has a high affinity for a 31bp sequence in the Yp1 gene necessary for normal steady state levels of Yp1 RNA in vivo has been identified.

Yolk proteins share a certain homologous domains with human low-density lipoproteins and human lipoprotein lipase.

Analysis of a Yp1-Adh promoter construct shows that separate regulatory sequences are required for female-specific Yp1 expression and ecdysone-inducible Yp1 expression in males.

A 125bp segment of DNA located 196bp upstream of the Yp1 cap site is sufficient to determine the sex-, stage- and fat body specific expression of the Yp1 gene. This region can confer specific expression patterns on a heterologous promoter, and acts in either orientation.

The sex-, time- and tissue-specific expression of the Yp1 and Yp2 genes, divergently transcribed and separated by 1225bp, indicates that there are two tissue-specifying elements acting on each gene. One is necessary for expression in fat body and the other for expression in the ovary.

Yp2 has been cloned and sequenced, and compared with Yp1. The intergenic region between Yp1 and Yp2 has also been sequenced.

Yp1 has been cloned.

The exon-intron structure of Yp1 has been determined.

The three major yolk proteins (encoded by Yp1, Yp2 and Yp3) have been isolated from mature eggs and characterised.

The levels of Yp1 protein in a variety of sex-transformation mutants (tra, dsx and tra2) have been studied.

Hormonal and genetic regulation of yolk formation has been reviewed.

Structural gene for the yolk protein YP1 found in recently-emerged female flies. Protein migrates at different rates in SDS-polyacrylamide gels when encoded by the electrophoretic variants Yp1^F (fast) and Yp1^S (slow), alleles that are female fertile and produce normal amounts of YP1. Yp1^ts1, which maps near the Yp1 locus and is believed to be an allele, produces a slow-migrating translation product that is present in reduced amounts in the hemolymph and the ovaries (Bownes and Hodson, 1980); this mutant is female sterile.