A new translational repression element and unusual transcriptional control regulate expression of don juan during Drosophila spermatogenesis.

The Drosophila don juan (dj) gene encodes a basic protein that is expressed solely in the male germline and shows structural similarities to the linker histone H1. Don Juan is located in two different subcellular structures: in the nucleus during the phase of chromatin condensation and later in the mitochondrial derivatives starting with spermatid individualization. The don juan gene is transcribed in primary spermatocytes under the control of 23 bp upstream in combination with downstream sequences. During meiotic stages and in early spermatid stages don juan mRNA is translationally repressed for several days. Analysis of male sterile mutants which fail to undergo meiosis shows that release of dj mRNA from translational repression is independent of meiosis. In gel retardation assays 60 nucleotides at the end of the dj leader form four major complexes with proteins that were extracted from testes but not with protein extracts from ovaries. Transformation studies prove that in vivo 35 bp within that region of the dj mRNA is essential to confer translational repression. UV cross-linking studies show that a 62 kDa protein specifically binds to the same region within the 5′ untranslated region. The dj translational repression element, TRE, is distinct from the translational control element, TCE, described earlier for all members of the Mst(3)CGP gene family. Moreover, expression studies in several male sterile mutants reveal that don juan mRNA is translated in earlier developmental stages during sperm morphogenesis than the Mst(3)CGP mRNAs. This proves that translational activation of dormant mRNAs in spermatogenesis occurs at different time-points which are characteristic for each gene, an essential feature for coordinated sperm morphogenesis.     

A negative transcriptional control element located upstream of the murine c-myc gene

[This corrects the article on p. 899 in vol. 5, PMID: 3013622.].     

Developmental control of the β-phaseolin gene requires positive, negative, and temporal seed-specific transcriptional regulatory elements and a negative element for stem and root expression

To identify cis-acting regulatory elements responsible for developmental control of the common bean seed storage protein β-phaseolin, a series of 5′-deletion mutants of the 782 bp upstream sequence together with the coding and 3′-regions of the β-phaseolin gene were used to transform tobacco. One major positive regulatory element (?295/?228) and a putative minimal promoter (?64/?14) were indicated by large reductions in phaseolin mRNA and protein concentrations in seeds of plants deficient for these regions. In addition, minor negative (?422/?296) and positive (?782/?423) elements also influenced the level of phaseolin mRNA expression in seeds. One temporal element (?295/?107) governed late expression of phaseolin mRNA in seeds, and possibly a second (?64/?14) regulated early expression. The ?64/?14 promoter containing two TATA boxes conferred spatially regulated gene expression, and was sufficient for a low level of expression in root and stem. Significant levels of phaseolin mRNA and protein were detected in stem cortices and secondary roots of plants lacking the ?295/?107 negative element. No significant expression in leaf tissue was detected. Results demonstrate that developmental control of β-phaseolin requires a minimal promoter, one element for the suppression of expression in root and stem tissue, three elements governing quantitative expression in seeds, and at least one temporal element controlling expression in seeds.     

Hormonal regulation of myosin heavy chain and alpha-actin gene expression in cultured fetal rat heart myocytes

Thyroid hormone regulates the expression of ventricular myosin isoenzymes by causing an accumulation of alpha-myosin heavy chain (MHC) mRNA and inhibiting expression of beta-MHC mRNA. However, the mechanism of thyroid hormone action has been difficult to examine in vivo because of its diverse actions. Accordingly, hormonal control of expression of six MHC isoform mRNAs and cardiac and skeletal alpha-actin mRNAs was studied in primary cultures of fetal rat heart myocytes grown in defined medium. The results indicate that in the absence of thyroid hormone, cultured heart cells express predominantly beta-MHC and cardiac alpha-actin mRNAs. Addition of 3,5,3'-triiodo-L-thyronine (T3) caused a rapid induction of alpha-MHC mRNA and decreased beta-MHC mRNA levels without affecting the skeletal muscle MHC mRNAs. There was an almost parallel change in the myosin isoenzymes. Cardiac alpha-actin mRNA levels were transiently increased by T3 treatment, but skeletal alpha-actin was unaffected. Elimination of insulin and epithelial growth factor from the medium did not alter the effects of T3 on cardiac MHC mRNA expression. Addition of various adrenergic agents to the medium had no appreciable effect on cardiac MHC mRNA expression despite the presence of functionally coupled alpha- and beta-adrenergic receptors. Addition of steroid hormones, muscarinic agents, and glucagon to the medium also had no effect. Thus, under defined conditions, T3 is able to regulate MHC gene expression at a pretranslational level without the need for other exogenous factors.     

High level expression of proteins using sequences from the ferritin heavy chain gene locus

An expression vector has been generated using a gene highly expressed under conditions found in a typical fed-batch bioreactor process. The ferritin heavy chain (HC) gene exhibits higher levels of expression in the late stages of a fed-batch bioreactor than in the early stages. This property was considered advantageous for an expression vector, since the maximal cell density would coincide with maximal expression. The rat ferritin HC genomic region was isolated and converted into an expression vector where large segments of 5' and 3' flanking regions were included in an attempt to recreate the same high level of expression in stably transfected cells. Expression from the resulting ferritin HC vector was compared to vectors containing the commonly used strong promoters, CMV IE, and SV40 early promoter/enhancer, in the generation of stable transfectants. The ferritin HC vector was able to generate cell lines with significantly higher expression levels than those under the control of the viral promoters.