An upstream regulatory region mediates high-level, tissue-specific expression of the human alpha 1(I) collagen gene in transgenic mice.

Studies in vitro have not adequately resolved the role of intronic and upstream elements in regulating expression of the alpha 1(I) collagen gene. To address this issue, we generated 12 separate lines of transgenic mice with alpha 1(I) collagen-human growth hormone (hGH) constructs containing different amounts of 5'-flanking sequence, with or without most of the first intron. Transgenes driven by 2.3 kb of alpha 1(I) 5'-flanking sequence, whether or not they contained the first intron, were expressed at a high level and in a tissue-specific manner in seven out of seven independent lines of transgenic mice. In most tissues, the transgene was expressed at levels approaching that of the endogenous alpha 1(I) gene and was regulated identically with the endogenous gene as animals aged. However, in lung, expression of the transgene was anomalously high, and in muscle, expression was lower than that of the endogenous gene, suggesting that in these tissues other regions of the gene may participate in directing appropriate expression. Five lines of mice were generated containing transgenes driven by 0.44 kb of alpha 1(I) 5'-flanking sequence (with or without the first intron), and expression was detected in four out of five of these lines. The level of expression of the 0.44-kb constructs in the major collagen-producing tissues was 15- to 500-fold lower than that observed with the longer 2.3-kb promoter. While transgenes containing the 0.44-kb promoter and the first intron retained a modest degree of tissue-specific expression, those without the first intron lacked tissue specificity and were poorly expressed in all tissues except lung. These results contribute to our understanding of the role of the first intron in regulating alpha1(I) gene expression and identify a region, upstream of the basal alpha1(I) promotor, which is necessary for full tissue-specific, developmentally regulated expression of the alpha1(I) collagen gene.     

Analysis of a genomic DNA segment carrying the wheat high-molecular-weight (HMW) glutenin Bx17 subunit and its use as an RFLP marker

Summary A genomic fragment containing the Bx17 high-molecular-weight (HMW) glutenin gene was isolated from a wheat genomic library. The fragment contains a coding region of 2.82kb with 1.98-kb downstream and 12.8-kb upstream flanking regions. The fragment was sequenced and compared with previously published glutenin genes from chromosomes 1A, 1B and 1D using a computer alignment package. The Bx17 gene shows marked similarity to the Bx7 gene sequence. A phenetic tree derived from the alignments is presented. Also shown are restriction fragment length polymorphisms (RFLPs) at the glutenin loci in a set of Australian and international wheat varieties using different regions of the glutenin clone as probes. The RFLPs correlated well with the protein composition in all cultivars analysed.     

Development and characterization of a chimaeric tissue-specific promoter in wheat and rice endosperm

The recently achieved significant improvement of cereal transformation protocols provides facilities to alter the protein composition of the endosperm, for example, to increase or decrease the quantity of one of its protein components or to express foreign molecules. To achieve this goal, strong endosperm-specific promoters have to be available. The aim of our work was to develop a more efficient tissue-specific promoter which is currently used. A chimaeric promoter was assembled using the 5′ UTR (1,900 bp) of the gene coding for the 1Bx17 HMW glutenin subunit protein, responsible for tissue-specific expression and the first intron of the rice actin gene (act1). The sequence around of the translation initial codon was optimized. The effect of the intron and promoter regulatory sequences, using different lengths of 1Bx17 HMW-GS promoter, were studied on the expression of uidA gene. The function of promoter elements, promoter length, and the first intron of the rice actin gene were tested by a transient expression assay in immature wheat endosperm and in stable transgenic rice plants. Results showed that insertion of the rice act1 first intron increased GUS expression by four times in transient assay. The shortest 1Bx17 HMW-GS promoter fragment (173 bp) linked to the intron and GUS reporter gene provided almost the same expression level than the intronless long 1Bx17 HMW-GS promoter. Analysis of the stable transformant plants revealed that 173 nucleotides were sufficient for endosperm-specific expression of the uidA gene, despite 13 nucleotides missing from the HMW enhancer sequence, a relevant regulatory element in the promoter region.     

Dissemination of the highly expressed Bx7 glutenin subunit (<Emphasis Type="Italic">Glu-B1al</Emphasis> allele) in wheat as revealed by novel PCR markers and RP-HPLC

Increased expression of the high molecular weight glutenin subunit (HMW-GS) Bx7 is associated with improved dough strength of wheat (Triticum aestivum L.) flour. Several cultivars and landraces of widely different genetic backgrounds from around the world have now been found to contain this so-called over-expressing allelic form of the Bx7 subunit encoded by Glu-B1al. Using three methods of identification, SDS-PAGE, RP-HPLC and PCR marker analysis, as well as pedigree information, we have traced the distribution and source of this allele from a Uruguayan landrace, Americano 44D, in the mid-nineteenth century. Results are supported by knowledge of the movement of wheat lines with migrants. All cultivars possessing the Glu-B1al allele can be identified by the following attributes: (1) the elution of the By sub-unit peak before the Dx sub-unit peak by RP-HPLC, (2) high expression levels of Bx7 (>39% Mol% Bx), (3) a 43 bp insertion in the matrix-attachment region (MAR) upstream of the gene promoter relative to Bx7 and an 18 bp nucleotide duplication in the coding region of the gene. Evidence is presented indicating that these 18 and 43 bp sequence insertions are not causal for the high expression levels of Bx7 as they were also found to be present in a small number of hexaploid species, including Chinese Spring, and species expressing Glu-B1ak and Glu-B1a alleles. In addition, these sequence inserts were found in different isolates of the tetraploid wheat, T. turgidum, indicating that these insertion/deletion events occurred prior to hexaploidization.     

Molecular characterization of high molecular weight glutenin subunit allele 1Bx23 in common wheat introduced from hexaploid triticale

High molecular weight glutenin subunit (HMW-GS) 1Bx23, an x-type subset encoded by Glu-B1p, which is only distributed in Triticum turgidum, was successfully transferred from hexaploid triticale to common wheat line SY95-71. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) shows that subunit 1Bx23 has a faster mobility than subunit 1Bx7 and 1Bx20, but slower than 1Bx17. Primers designed from the conserved regions in wheat HMW-GS gene promoter and coding sequences were used to amplify the genomic DNA of SY95-71. Total nucleotide sequences of 3426 bp including an open reading frame of 2385 bp and upstream sequence of 1038 bp were obtained. Compared with the reported gene sequences of Glu-B1-1 alleles, including 1Bx7, 1Bx14, 1Bx20 and 1Bx17, the promoter region of the 1Bx23 was displayed close to 1Bx7 and 1Bx17. The deduced amino acid sequence of coding region of 1Bx23 exhibited 34, 30, 20 and 22 amino acid substitutions from that of 1Bx14, 1Bx20, 1Bx7 and 1Bx17, respectively. A phylogenetic tree based on the nucleotide sequence alignment of the Glu-1Bx alleles shows that the 1Bx23 are apparently clustered with 1Bx7 and 1Bx17, and more ancient than 1Bx14 and 1Bx20, suggesting that the evolution speeds are different among Glu-1Bx genes. Additionally, the potential use of wheat line SY95-71 to further screen the quality contribution of unique subunit 1Bx23 is also discussed.     

The cyclic nucleotide phosphodiesterase gene of Dictyostelium discoideum contains three promoters specific for growth, aggregation, and late development.

The cyclic nucleotide phosphodiesterase (phosphodiesterase) plays essential roles throughout the development of Dictyostelium discoideum. It is crucial to cellular aggregation and to postaggregation morphogenesis. The phosphodiesterase gene is transcribed into three mRNAs, containing the same coding sequence connected to different 5' untranslated sequences, that accumulate at different times during the life cycle. A 1.9-kilobase (kb) mRNA is specific for growth, a 2.4-kb mRNA is specific for aggregation, and a 2.2-kb mRNA is specific for late development and is only expressed in prestalk cells. Hybridization of RNA isolated from cells at various stages of development with different upstream regions of the gene indicated separate promoters for each of the three mRNAs. The existence of specific promoters was confirmed by fusing the three putative promoter regions to the chloramphenicol acetyltransferase reporter gene, and the analysis of transformants containing these constructs. The three promoters are scattered within a 4.1-kilobase pair (kbp) region upstream of the initiation codon. The late promoter is proximal to the coding sequence, the growth-specific promoter has an initiation site that is 1.9 kbp upstream of the ATG codon, and the aggregation-specific promoter has an initiation site 3 kbp upstream.     

Distal Sox binding elements of the alphaB-crystallin gene show lens enhancer activity in transgenic mouse embryos

alphaB-Crystallin, a member of the small heat shock protein (sHSP) family, is expressed in various tissues including lens, heart, and skeletal muscle. Previously we identified the gene of HSPB2, another member of the sHSP family, located 1-kb upstream of the alphaB-crystallin gene in a head-to-head manner. In the present study, we found a highly conserved region of 220 bp approximately 2.4-kb upstream of the alphaB-crystallin gene and examined its role in expression of the alphaB-crystallin gene. Transgenic mice containing 3 kb of the upstream sequence of the alphaB-crystallin gene showed lacZ reporter gene expression in the lens as well as the myotome and heart on embryonic day 12.5. Deletion analysis revealed that the -2656/-2267 region including the conserved region with four putative Sox binding elements (E1-E4) exhibits lens enhancer activity toward the alphaB-crystallin promoter. Gel shift assays showed that the Sox1 and Sox2 proteins preferentially bound to E2 and E4. Moreover, disruption of E2 and E4 abolished the reporter gene expression in the lens. These results indicate that the newly identified enhancer with Sox elements activates the alphaB-crystallin promoter in the lens, although they are separated by the entire HSPB2 gene.     

Proximal upstream region of zebrafish bone morphogenetic protein 4 promoter directs heart expression of green fluorescent protein

We examined the activity of the bone morphogenetic protein 4 (BMP4) promoter in zebrafish embryos via transient and stable transgenic expression analyses in order to obtain a better understanding of the regulation of BMP4 tissue-specific expression. Transient expression studies showed that the 9.0-kb BMP4 promoter/upstream region drove green fluorescent protein (GFP) expression mainly in the heart. Deletion analyses indicated the existence of multiple regulatory elements in the 7.5-kb BMP4 promoter/proximal upstream region. In addition, a coinjection experiment further demonstrated the 2.4-kb Bgl II-Hind III DNA region contains major positive regulatory elements. In addition, stable transgenic lines were established to further confirm the heart-specificity of this segment in BMP4 promoter. The results showed that GFP was mainly localized in the myocardium of developing ventricles of 48-hpf (hours postfertilization), 72-hpf, and 100-hpf transgenic F(1) embryos. Together, these results indicate that the 7.5-kb BMP4 promoter/proximal upstream region specifically contains regulatory elements for BMP4 expression in the heart, while regulatory elements for other endogenous BMP4-expressing tissues may reside in more distal regions and/or in introns.     

Differential regulation of the rat phosphoenolpyruvate carboxykinase gene expression in several tissues of transgenic mice.

The selective expression of a unique copy gene in several mammalian tissues has been approached by studying the regulatory sequences needed to control expression of the rat phosphoenolpyruvate carboxykinase (PEPCK) gene in transgenic mice. A transgene containing the entire PEPCK gene, including 2.2 kb of the 5'-flanking region and 0.5 kb of the 3'-flanking region, exhibits tissue-specific expression in the liver, kidney, and adipose tissue, as well as the hormonal and developmental regulation inherent to endogenous gene expression. Deletions of the 5'-flanking region of the gene have shown the need for sequences downstream of position -540 of the PEPCK gene for expression in the liver and sequences downstream of position -362 for expression in the kidney. Additional sequences upstream of position -540 (up to -2200) are required for expression in adipose tissue. In addition, the region containing the glucocorticoid-responsive elements of the gene used by the kidney was identified. This same sequence was found to be needed specifically for developmental regulation of gene expression in the kidney and, together with upstream sequences, in the intestine. The apparently distinct sequence requirements in the various tissues indicate that the tissues use different mechanisms for expression of the same gene.