Sheep α-globin gene sequences: Implications for their concerted evolution and for the down-regulation of the 3′ genes

In sheep as in man and most other mammals, there are two -globin genes (^I and ^II), which are expressed at different levels, the upstream gene being the most efficient. In -globin gene triplication and quadruplication, this trend is confirmed, i.e., the -chain output of the downstream genes progressively decreases. In this study, we have determined the complete sequence of the cDNAs and of both the introns in a triple- haplotype in which each gene could be recognized for the presence of distinct alleles. The sequence analysis reveals that the bodies of the three -globin genes are essentially identical (99.9% homology) and moreover indicates that the down-regulation of additional -globin genes in sheep is not the effect of sequence variation from the Cap to the Poly(A) addition sites. This striking similarity among -genes is higher than that seen in other mammals and is probably sustained by particularly efficient mechanisms of gene conversion and cross-over fixation. Correspondence to: Dr. M.S. Ristaldi     

Transient expression of the β-glucuronidase gene in embryogenic callus of Picea mariana following microprojection

A microprojection protocol using the DuPont Biolistic particle delivery system and the -glucuronidase (GUS) reporter gene fused with the 35S promoter of Cauliflower mosaic virus (CaMV) was developed for Picea mariana callus. Comparison of four tungsten microprojectile sizes showed the highest transient gene expression with 1.11m diameter particles. Adsorption of DNA on the microcarriers using calcium chloride led to higher GUS gene activity than using polyethylene glycol. GUS gene activity in P. mariana was the highest when cells were treated 5 and 6 days after subculturing to fresh media. The wheat ABA-inducible Em gene promoter yielded 4.5 times higher GUS gene activity than the 35S CaMV promoter. Comparison of transient GUS gene expression among 10 P. mariana embryogenic cell lines from six different open-pollinated families showed comparable gene activity, with the exception of one family showing no GUS gene activity.     

Transient expression of the β-glucuronidase gene in tissues ofArabidopsis thaliana by bombardment-mediated transformation

Particle bombardment has proved to be useful for the transformation of plants. We have previously reported successful transient expression of the beta-glucuronidase (GUS) gene in cultured plant cells and tissues and the stable transformation of various plants using a pneumatic particle gun. In this chapter, we describe transient expression of the GUS gene in Arabidopsis thaliana leaves and roots using the pneumatic particle gun.     

Transient expression of the β-glucuronidase gene introduced into barley coleoptile cells by microinjection

A -glucuronidase gene was introduced directly into barley (Hordeum vulgare L. cv. Kobinkatagi) coleoptile cells by microinjection and transient expression of the gene was examined. Inner epidermis tissue of coleoptiles was excised and injected with plasmid DNA, pBI221, carrying cauliflower mosaic virus 35S promoter, -glucuronidase gene, and a nopaline synthase polyadenylation region. Histochemical assay for -glucuronidase production showed positive enzyme activity only in coleoptile cells injected with plasmid DNA. Expression of the -glucuronidase gene was examined chronologically using honogenates of injected coleoptile tissues. Glucuronidase activity first appeared after 6 hr, reached the maximum level 24 hr after injection, and decreased afterwards. These results suggest that microinjection of coleoptile tissues may be a useful approach for the genetic engineering of Gramineae plants in which protoplast regeneration is difficult.     

Sequence analysis of the 5′-untranslated region of the human H1 histamine receptor-encoding gene

A clone containing the H₁ histamine receptor (H₁HR)-encoding gene was isolated from a human genomic DNA library. The 5′-UTR of the H₁HR gene reported here differs upstream from bp −142 from that reported previously [Fukui et al., Biochem. Biophys. Res. Comm. 201 (1994) 894–901]. PCR amplification utilizing primer pairs derived from the 5′-UTR reported herein amplified a DNA fragment of the expected size from human genomic DNA whereas 5′-UTR primers derived from the Fukui et al. sequence did not yield a PCR product. The 5′-UTR of H₁HR contains potential TATA and CCAAT boxes, a CACCC sequence, potential GREs and other DNA-binding motifs.     

Secondary embryogenesis and transient expression of the β-glucuronidase gene in hypocotyls of rapeseed microspore-derived embryos

Secondary embryogenesis from rapeseed microspore-derived embryos (MDEs) was studied in three Brassica napus L. cultivars Global, PF₇₀₄ and Option. The best results in terms of secondary embryogenesis percentage obtained in cultures of Global and PF₇₀₄ MDEs (75.88 and 65.97 %, respectively) and PF₇₀₄ produced the highest number of secondary embryos per each primary embryo (14.91 ± 2.18). After optimization of physical parameters, rapeseed hypocotyls of MDEs were bombarded with microcarriers coated with a plasmid containing GUS reporter gene. The highest levels of transient GUS expression were obtained using bombardment with gold particles of 1.6 μm, at helium pressure of 9.3 MPa, a bombardment distance of 9 cm, chamber vacuum pressure of 7.1 × 10⁻⁶ kPa and single bombardment in bombardment medium containing 0.4 M mannitol.     

Intron 2 of human beta-globin in 3′-untranslated region enhances expression of chimeric genes

The possibility of enhancing heterologous gene expression in mammalian cells by the introduction of an intron in 3′ untranslated region (UTR) was investigated. To this end, a fragment of human betaglobin gene with intron 2 and flanked exon regions was introduced into the vector-encoding green fluorescent protein TagGFP2 after the TagGFP2 stop-codon (Int⁺). The distance between the stop-codon and the exon junction was 35 nucleotides. It ensured that Int+ mRNA was resistant to degradation by nonsense mediated decay (NMD) machinery. A control vector Intcontained corresponding intronless sequence of the beta-globin mRNA. On the same plasmid, the second gene encoded far-red fluorescent protein Katushka was used to normalize fluorescence for transfection efficiency and expression level in individual cells. Transiently transfected HEK293T cells were analysed by flow cytometry. It was shown that cells transfected with plasmid carrying the Int+ gene possess 1.8 ± 0.2 fold higher green fluorescence compared to Intcells. The observed effect was used to enhance expression of destabilized variants of yellow fluorescent protein TurboYFP-dest with high degradation rate in mammalian cells. We believe that introduction of beta-globin intron in the 3′-UTR of the chimeric gene can be used to enhance its expression and may be advantageous in some cases when usage of 5′ UTR intron is inappropriate.     

Promoter sequences from two different Brassica napus tapetal oleosin-like genes direct tapetal expression of β-glucuronidase in transgenic Brassica plants

To investigate the sequences responsible for the regulated expression of tapetal-specific oleosin-like genes, ca. 2 kb of the 5-upstream regions from two divergent genes, OlnB;4 and OlnB;13, were isolated, sequenced and fused to the reporter gene -glucuronidase for study in transgenic Brassica napus plants. Although the proteins encoded by these two genes are highly divergent, except for the conserved oleosin-like domain, the first 250 bp of their 5-upstream regions was 86% identical, including a region of 150 bp upstream from the TATA box. Analysis of 42 independent transformants by histochemical and fluorometric methods showed that both promoters directed tapetal-specific expression that peaked at the 4 mm flower bud stage.     

DNA variants within the 5′-flanking region of milk-protein-encoding genes II. The β-lactoglobulin-encoding gene

For the detection of polymorphisms within the 5-flanking region of the -lactoglobulin (-LG) -encoding gene a nucleotide sequence containing 795 bp of the promoter and 59 bp of exon I was cloned and sequenced. After comparing the sequence from the DNA of 11 diverse cows (different breeds and milk-protein yields), 14 singlebp substitutions were identified within the 5-flanking region and two in the 5-untranslated region (5-UTR) of exon I. Some of the variants are located in potential binding sites for trans-acting factors or in the 5-UTR. A PCR-based RFLP analysis was performed, and the genotypes of an additional 60 cows were identified at five variable 5-flanking sites. The results reveal three frequent combinations between the A and B alleles of the protein-coding region and the novel 5-flanking DNA variants. This finding may explain the differences of the protein-variant-dependent -LG synthesis (A>B) observed in vivo. A sequence comparison of the bovine and ovine promoters reveals an homology of 92.8% and shows a higher degree of conservation between positions -600 and -300.