Developmental changes in proteins of purified membranes of chicken lenses and evidence for contamination by cytoplasmic δ-crystallin

Urea-washed membranes from embryonic chick lenses (15 days old) and from the cortical and nuclear regions of adult chicken lenses (1 year) have been prepared by repeated centrifugation through discontinuous density gradients. The protein components of the isolated membranes have been examined by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate and urea. Proteins with molecular weights of 75 000, 56 000, 54 000, 48 000, 34 000, 32 000, 25 000, and 22 000 were present in all the membrane preparations, although their proportions changed during development. One additional protein, molecular weight 70 000, was seen only in the embryonic lens membranes. The greatest developmental change was the increase in 25 000 molecular weight protein from 12% in the embryonic lens to about 45% in the adult lens. Since it has been suggested that this protein is associated with gap junctions, its increase during development may reflect a corresponding increase in the number of gap junctions in the lens.The 50 000 molecular weight protein of embryonic lens membranes and membranes of adult nuclear lens fibers consisted at least partly of δ-crystallin, since δ-crystallin peptides could be identified in tryptic pepetide maps of the isolated protein after in vitro radioiodination. Peptide maps of the 50 000 molecular weight protein of cortical lens fiber membranes contained no identifiable δ-crystallin peptides, although it is possible that modified δ-crystallin peptides may be present. The level of cytoplasmic contamination of the membrane fraction was estimated by preparing lens membranes in the presence of added δ-[³⁵S]crystallin. The results indicated that cytoplasmic contamination contributes significantly to the presence of δ-crystallin in lens membrane preparations.     

Degradation of δ-crystallin mRNA in the lens fiber cells of the chicken

δ-Crystallin is the principal protein synthesized in the embryonic chicken lens. After hatching δ-crystallin synthesis decreases and eventually ceases. We have determined when the δ-crystallin messenger RNA (mRNA) disappears from the lens fiber cells during the first year of age by cell-free translation of lens RNA in a reticulocyte lysate, RNA blot (Northern) hybridization, and in situ hybridization. The hybridization was performed with a nick-translated, cloned δ-crystallin cDNA (pδCr2). δ-Crystallin mRNA was present in the lens until 3 months of age and disappeared between the third and fifth month after hatching. The in situ hybridization experiments indicated that the δ-crystallin mRNA was present throughout the lens fiber mass until 1 month after hatching and was greatly reduced in the cortical fiber cells thereafter. In contrast to earlier stages, then, the cortical fiber cells differentiating at the lens equator after about 1 month of age do not accumulate δ-crystallin mRNA. The data also indicate that the maximal half-life of functional δ-crystallin mRNA in the posthatched chicken lens is about 2 months.     

Functional analysis of the chicken PPARγ gene 5'-flanking region and C/EBPα-mediated gene regulation

Peroxisome proliferator-activated receptor-γ (PPARγ) and CCAAT/enhancer binding protein-α (C/EBPα) are the master regulators of adipogenesis. The regulatory mechanism of PPARγ and C/EBPα gene expression is clear in mammals, however, little is known in chicken. The aim of the present study was to characterize chicken PPARγ promoter and investigate whether PPARγ could be regulated by C/EBPα in chickens. A 2-kb nucleotide sequence upstream of the start codon of chicken PPARγ gene was cloned and characterized by using bioinformatics and experimental approaches. This 2-kb promoter region exhibited strong promoter activity in DF1 cells. The reporter gene assay showed that the chicken C/EBPα could activate PPARγ gene promoter. Further study by electrophoretic mobility shift assay and mutational analysis revealed that the chicken C/EBPα could directly bind to and regulate the PPARγ gene promoter. Our results demonstrate that PPARγ can be directly regulated by C/EBPα in chickens.     

Conservation of δ-crystallin gene structure between ducks and chickens

A cloned chicken delta-crystallin cDNA was used to identify two putative delta-crystallin genes in the duck by Southern blot hybridization. A DNA fragment containing most of one of these genes was isolated from a library made in bacteriophage lambda Charon 28A containing genomic DNA from 14-day-old embryonic ducks. Electron microscopy, partial gene sequencing, primer extension analysis using duck mRNA, and comparison with the well-characterized chicken delta-crystallin genes suggest that our cloned duck delta-crystallin gene, like the chicken delta-crystallin genes, is 8-10 kb long and contains 17 exons. Hybridization and sequencing data show great similarity between the homologous 5' untranslated and coding exons of the duck and chicken delta-crystallin genes. Overall, the homologous introns also appear to have approximately 30% sequence similarity, and have been subject to deletion/insertion events. Our partial characterization of duck delta-crystallin gene sequences suggests that this avian and reptilian crystallin family has been conserved during evolution, as have the other crystallin gene families that are expressed in the eye lens.     

The control of δ-crystallin gene expression during lens cell development: Dissociation of cell elongation,cell division, δ-crystallin synthesis,and δ-crystallin mRNA accumulation

Previous studies have shown that freshly explanted 6-day-old embryonic chick lens epithelial cells elongate, differentially increase their synthesis of δ-crystallin, and accumulate δ-crystallin mRNA when cultured with fetal calf serum; in contrast, precultured serum-starved 6-day-old and freshly explanted 19-day-old embryonic epithelial cells divide when treated with fetal calf serum. We have explored whether the stimulation of δ-crystallin gene expression (as measured by δ-crystallin synthesis and δ-crystallin mRNA accumulation) is affected by inhibiting lens cell elongation with colchicine, and whether δ-crystallin gene expression is increased in lens epithelial cells stimulated to divide by treatment with fetal calf serum, as it is in those stimulated to elongate by treatment with serum. Three new findings were made in this study. First, the stimulation of δ-crystallin gene expression does not require elongation of the cultured lens cells. Second, a decreased proportion of δ-crystallin synthesis is observed in lens epithelial cells during normal development and during serum starvation; in neither case is this decrease associated with a reduction in the number of δ-crystallin mRNA sequences per cell. Finally, serum stimulation of lens cell division does not increase the proportion of δ-crystallin synthesis, but can promote the accumulation of δ-crystallin mRNA. Thus, the relative proportion of δ-crystallin synthesized during chick lens development is not solely a function of the number of δ-crystallin mRNA sequences in the lens cells.     

Enzymic synthesis of argininosuccinate catalyzed by δ-crystallin

Summary A fast and practical procedure has been developed for the synthesis of argininosuccinate using immobilized duck lens -crystallin as catalyst.     

Detailed analysis of the δ-crystallin mRNA-expressing region in early development of the chick pituitary gland

Although δ-crystallin (δ-crys), also known as lens protein, is transiently expressed in Rathke's pouch (RP) of the chick embryo, detailed temporal and spatial expression patterns have been obscure. In this study, to understand the relationship between the δ-crys mRNA-expressing region and RP formation, we examined the embryonic expression pattern of δ-crys mRNA in the primordium of the adenohypophysis. δ-crys mRNA expression was initially found at stage 15 anterior to the foregut and posterior to the invaginated oral ectoderm. After RP formation, the δ-crys mRNA was expressed in the post-ventral region of RP and the anterior region of RP. δ-crys mRNA expression was then restricted to the cephalic lobe of the pituitary gland. From stage 20, the δ-crys and alpha-glycoprotein subunit (αGSU) mRNA-expressing regions were almost completely overlapping. The αGSU mRNA-expressing region is thought to be the primordium of the pars tuberalis, and these regions were overlapped with the Lhx3 mRNA-expressing region. The intensity of δ-crys mRNA expression gradually decreased with development and completely disappeared by stage 34. These results suggest that the embryonic chick pituitary gland consists of two different regions labeled with δ-crys and Lhx3.     

Developmental regulation of eukaryotic gene loci: which cis-regulatory information is required?

It is becoming increasingly accepted that gene loci comprise an extensive cis-regulatory system that encodes different layers of regulatory information, all of which are necessary to achieve and maintain tissue-specific gene expression in ontogeny. To gain a detailed understanding of developmental processes, it is clearly necessary to unravel the molecular basis behind the different regulatory processes that control gene expression. This information is also of utmost importance for any practical application that uses gene transfer technology.     

Cloning and functional analysis of the Gβ gene Mgb1 and the Gγ gene Mgg1 in Monascus ruber

The ascomycetous fungus Monascus ruber is one of the most well-known species widely used to produce Monascus-fermentation products for natural food colorants and medicine. Our previous research on the Gα subunit Mga1 and the regulator of G protein signaling MrflbA indicated that heterotrimeric G protein signaling pathways were involved in aspects of growth, sporulation and secondary metabolite production in M. ruber. To better understand the G protein signaling pathways in this fungus, a Gβ subunit gene (Mgb1) and a GΓ subunit gene (Mgg1) were cloned and investigated in the current study. The predicted Mgb1 protein consisted of 353 amino acids and Mgg1 consisted of 94 amino acids, sharing marked similarity with Aspergillus Gβ and GΓ subunits, respectively. Targeted deletion (Δ) of Mgb1 or Mgg1 resulted in phenotypic alterations similar to those resulting from ΔMga1, i.e., restricted vegetative growth, lowered asexual sporulation, impaired cleistothecial formation, and enhanced citrinin and pigment production. Moreover, deletion of Mgg1 suppressed the defects in asexual development and in biosynthesis of citrinin and pigment caused by the absence of MrflbA function. These results provide evidence that Mgb1 and Mgg1 form a functional GβΓ dimer and the dimer interacts with Mga1 to mediate signaling pathways, which are negatively controlled by MrflbA, for growth, reproduction and citrinin and pigment biosynthesis in M. ruber.     

Developmental regulation of the maize Zm-p60.1 gene encoding a β-glucosidase located to plastids

A β-glucosidase that cleaves the biologically inactive hormone conjugates cytokinin-O- and kinetin-N3-glucosides is encoded by the maize Zm-p60.1 gene. The expression of the Zm-p60.1 gene was analyzed by Northern blot analysis and in-situ hybridization. It was found that the expression levels of the Zm-p60.1-specific mRNA changed after pollination of carpellate inflorescences. The Zm-p60.1 cDNA was expressed in E. coli and antibodies were raised against this protein. An antibody was used to determine the tissue-specific localization of this protein. By in situ immunolocalization experiments, this protein was found to be located in cell layers below the epidermis and around the vascular bundles of the coleoptile. In the primary leaf, the Zm-p60.1 protein was detected in cells of the outermost cell layer and around the vascular tissue. In floral tissue, Zm-p60.1 was present in the glumes, the carpels and in the outer cell layer of the style. In coleoptiles, as determined by immuno-electronmicroscopy, the Zm-p60.1 protein was located exclusively in the plastids. Received: 11 August 1998 / Accepted: 30 December 1998     

Control of yeast cell type by the mating type locus: Positive regulation of the α-specific STE3 gene by the MATα 1 product

The mating type locus (MAT) determines the three yeast cell types, a, α, and a/α. It has been proposed that alleles of this locus, MATa and MATα, encode regulators that control expression of unlinked genes necessary for mating and sporulation. Specifically, the α1 product of MATα is proposed to be a positive regulator of α-specific genes. To test this view, we have assayed RNA production from the α-specific STE3 gene in the three cell types and in mutants defective in MATα. The STE3 gene was cloned by screening a yeast genomic clone bank for plasmids that complement the mating defect of ste3 mutants. Using the cloned STE3 gene as a probe, we find that a cells produce STE3 RNA, whereas a and a/a cells do not. Furthermore, matα 1 mutants do not produce STE3 RNA, whereas matα 2 mutants do. These results show that the STE3 gene, required for mating only by α cells, is expressed only in α cells. They show also that production of RNA from the STE3 gene requires the α1 product of MATα. Thus α1 positively regulates at least one α-specific gene by increasing the level of that gene's RNA product.     

The regulation of the UCH-L1 gene by transcription factor NF-κB in podocytes

In kidney, the ubiquitin carboxy-terminal hydrolase 1 (UCH-L1) is involved in podocyte injury and proteinuria but details of the mechanism underlying its regulation are not known. Activation of NF-κB is thought to be the predominant risk factor for kidney disease; therefore, it is postulated that UCH-L1 may be one of the NF-κB target genes. In this study, we investigated the involvement of NF-κB activation in the regulation of UCH-L1 expression and the function of murine podocytes. Stimulation of podocytes with the cytokines TNF-α and IL-1β up-regulated UCH-L1 expression rapidly at the mRNA and protein levels and the NF-κB-specific inhibitor pyrrolidine dithiocarbamate resulted in down-regulation. NF-κB up-regulates UCH-L1 via binding the ? 300 bp and ? 109 bp sites of its promoter, which was confirmed by the electrophoretic mobility shift assay of DNA–nuclear protein binding. In the renal biopsy from lupus nephritis patients, the expressions of NF-κB and UCH-L1 increased in immunohistochestry staining and were positively correlated. Activation of NF-κB up-regulates UCH-L1 expression following changing of other podocytes molecules, such as nephrin and snail. These results suggest that activation of the NF-κB signaling pathway could be the major pathogenesis to up-regulate UCH-L1 in podocyte injury, followed by the turnover of other molecules, which might result in morphological changes and dysfunction of podocytes. This work help us to understand the effect of NF-κB on specific target molecules of podocytes, and suggest that targeting the NF-κB–UCH-L1 interaction could be a novel therapeutic strategy for the treatment of podocyte lesions and proteinuria.     

Phylogenetic analysis of the Glomeromycota by partial β-tubulin gene sequences

The 3’ end of the β-tubulin gene was amplified from 50 isolates of 45 species in Glomeromycota. The analyses included a representative selection of all families except Pacisporaceae and Geosiphonaceae. Phylogenetic analyses excluded three intron regions at the same relative positions in all species due to sequence and length polymorphisms. The β-tubulin gene phylogeny was similar to the 18S rRNA gene phylogeny at the family and species level, but it was not concordant at the order level. Species in Gigasporaceae and Glomeraceae grouped together but without statistical support. Paralogous sequences in Glomus species likely contributed to phylogenetic ambiguity. Trees generated using different fungal phyla as out-groups yielded a concordant topology. Family relationships within the Glomeromycota did not change regardless if the third codon position was included or excluded from the analysis. Multiple clones from three isolates of Scutellospora heterogama yielded divergent sequences. However, phylogenetic patterns suggested that only a single copy of the β-tubulin gene was present, with variation attributed to intraspecific sequence divergence.