Male Gametic Cell-specific Histone <Emphasis Type="Italic">gH2A</Emphasis> Gene of <Emphasis Type="Italic">Lilium longiflorum</Emphasis>: Genomic Structure and Promoter Activity in the Generative Cell

A genomic clone containing the gH2A gene, a histone variant specifically expressed in male gametic cells within the pollen of Lilium longiflorum, was isolated. Sequence analysis revealed that the coding region of the gene is interrupted by one intron, as is the case with the somatic type of plant histone H2A genes, suggesting derivation from the same ancestral gene containing one intron. In addition, a 2.8-kbp fragment of the 5′ upstream region of gH2A contained TATA and CAAT boxes, but neither a plant histone-specific regulatory DNA element nor vegetative cell-specific cis-elements were found. A histochemical study of stable transformants demonstrated that the 5′ upstream region of the gene can drive gene expression specifically in the generative cell of pollen; no activity was detectable in the vegetative cell or in other reproductive and vegetative tissues of transgenic Nicotiana tabacum. These results strongly suggest that the generative cell can direct specific gene expression, that this expression may be regulated by a putative male gametic factor, and that the gH2A promoter may therefore serve as a useful male gametic cell fate marker in angiosperms.     

Construction of a binary transgenic gene expression system for recombinant protein production in the middle silk gland of the silkworm <Emphasis Type="Italic">Bombyx mori</Emphasis>

To construct an efficient system for the production of recombinant proteins in silkworm (Bombyx mori), we investigated the promoter activity of the silkworm sericin 1, 2, and 3 genes (Ser1, Ser2, and Ser3) using a GAL4/UAS binary gene expression system in transgenic silkworm. The promoter activity of the upstream region of Ser1 was strong, yielding high expression of an enhanced green fluorescent protein (EGFP) transgene in the middle and posterior regions of the middle silk gland (MSG) after day 2 of the fifth instar. The Ser3 upstream region exhibited moderate promoter activity in the anterior MSG, but the Ser2 upstream region did not exhibit any promoter activity. Since the strongest promoter activity was observed for Ser1, we devised a system for the production of recombinant proteins using a GAL4–Ser1 promoter construct (Ser1-GAL4). Transgenic silkworms harboring both the Ser1-GAL4 construct and the previously reported upstream activating sequence (UAS)–EGFP construct, which contains the TATA box region of the Drosophila hsp70 gene, yielded approximately 100 μg EGFP per larva. When we then analyzed the TATA box region, signal peptide, and intron sequences for their effects on production from the UAS-EGFP construct, we found that the optimization of these sequences effectively increased production to an average of 500 μg EGFP protein per transgenic larva. We conclude that this binary system is a useful tool for the mass production of recombinant proteins of biomedical and pharmaceutical interest in silkworm.     

Functional characterization of a chicken major histocompatibility complex class II B gene promoter

 A 0.7 kilobase (kb) DNA fragment from the 5′ flanking region of a chicken major histocompatibility complex (MHC) class II B gene was cloned into chloramphenicol acetyltransferase (CAT) reporter vectors and was transfected into a chicken macrophage cell line that expresses a low level of MHC class II antigens. Positive orientation-dependent promoter activity of the chicken DNA was evident in a reporter construct containing an SV40 enhancer. Deletion analysis of this 0.7 kb DNA fragment revealed a short fragment in the 3′ end that was crucial for the promoter function and negative regulatory elements (NRE) located further upstream. The conserved MHC class II X and Y boxes did not have a significant effect on promoter activity. Sequence analysis of the 0.7 kb class II B gene upstream region suggests possible involvement of interferon (IFN), E twenty-six specific (ETS)-related proteins, and other factors in regulating this promoter. A chicken T-cell line culture supernatant increased surface expression of MHC class II antigens, as well as class II promoter activity, in this macrophage cell line. This first functional characterization of a chicken MHC class II B gene promoter will aid in understanding the regulatory mechanisms that control the expression of these genes. Received: 9 July 1996 / Revised: 7 October 1996     

Structure and regulation of the dnaA promoter region in three Streptomyces species

 The regulatory region of the Streptomyces dnaA gene comprises a single promoter and two DnaA boxes that are located upstream of the promoter. Comparative analysis of the dnaA promoter region from S. chrysomallus, S. lividans and S. reticuli revealed that the location, spacing and orientation of the DnaA boxes are conserved. In vitro studies demonstrated that efficient binding of the Streptomyces DnaA protein to DNA requires the presence of two DnaA boxes. In vivo analysis of dnaA promoter mutants deleted for one or both DnaA boxes indicated that the dnaA gene is autoregulated. However, the degree of derepression observed is relatively modest. Received: 12 July 1999 / Accepted: 19 October 1999     

Abscisic acid-regulated Glb1 transient expression in cultured maize P3377 cells

In a study of the 5′-flanking sequence of the Zea mays L. (maize) Glb1 gene in vitro, serial promoter deletions were generated and linked with the β-glucuronidase (GUS) reporter gene. The promoter deletion-GUS fusions were introduced into the maize P3377 cell line by particle bombardment. GUS assays indicated that treatment of the maize cultured cells with abscisic acid (ABA) was required for Glb1-driven GUS transient expression, and that the –272-bp sequence of the Glb1 promoter was sufficient for ABA-regulated expression of GUS. The longest undeleted sequence used, –1391 GUS, showed relatively low expression which could be indicative of an upstream silencer element in the Glb1 promoter between –1391 and –805. Further studies show that the Glb1-driven GUS activity of bombarded maize P3377 cells increases with increasing ABA concentration (up to 100–300 μm). Site-directed mutagenesis of a putative ABA response element, Em1a, abolished GUS expression in P3377 cells. This observation indicated that the Em1a sequence in the Glb1 5′ regulatory region is responsible for the positive ABA regulation of gene expression. Received: 9 May 1997 / Revision received: 9 November 1997 / Accepted: 8 December 1997     

Homology Modeling Based Solution Structure of Hoxc8-DNA Complex: Role of Context Bases Outside TAAT Stretch

Abstract

The 3D structure of neither Hoxc8 nor Hoxc8-DNA complex is known. The repressor protein Hoxc8 binds to the TAAT stretch of the promoter of the osteopontin gene and modulates its expression. Over expression of the osteopontin gene is related to diseases like osteoporosis, multiple sclerosis, cancer et cetera. In this paper we have proposed a 3D structure of Hoxc8-DNA complex obtained by Homology modeling and molecular dynamics (MD) simulation in explicit water. The crystal structure (9ant.pdb) of Antennapedia homeodomain in complex with its DNA sequence was chosen as the template based on (i) high sequence identity (85% for the protein and 60% for the DNA) and (ii) the presence of the TAAT stretch in interaction with the protein. The resulting model was refined by MD simulation for 2.0ns in explicit water. This refined model was then characterized in terms of the structural and the interactional features to improve our understanding of the mechanism of Hoxc8-DNA recognition. The interaction pattern shows that the residues Ile¹⁹⁵, Gln¹⁹⁸, and Asn¹⁹⁹, and the bases S2-⁴TAATG⁸ are most important for recognition suggesting the stretch TAATG as the ‘true recognition element’ in the present case. A strong and long-lived water bridge connecting Gln¹⁹⁸ and the base of S1-C⁷ complementary to S2-G⁸ was observed. Our predicted model of Hoxc8-DNA complex provides us with features that are consistent with the available experimental data on Hoxc8 and the general features of other homeodomain-DNA complexes. The predictions based on the model are also amenable to experimental verification.     

Characterization of Upstream Sequences of the <Emphasis Type="Italic">LOJ</Emphasis> Gene Leads to Identification of a Novel Enhancer Element Conferring Lateral Organ Junction-Specific Expression in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Isolation and characterization of promoters are important in understanding gene regulation and genetic engineering of crop plants. Earlier, a pentatricopeptide repeat protein (PPR) encoding gene (At2g39230), designated as Lateral Organ Junction (LOJ) gene, was identified through T-DNA promoter trapping in Arabidopsis thaliana. The upstream sequence of the LOJ gene conferred on the reporter gene a novel LOJ-specific expression. The present study was aimed at identifying and characterizing the cis-regulatory motifs responsible for tissue-specific expression in the −673 and +90 bases upstream of the LOJ gene recognized as LOJ promoter. In silico analysis of the LOJ promoter revealed the presence of a few relevant regulatory motifs and a unique feature like AT-rich inverted repeat. Deletion analysis of the LOJ promoter confirmed the presence of an enhancer-like element in the distal region (−673/−214), which stimulates a minimal promoter-like sequence in the −424/−214 region in a position and orientation autonomous manner. The −136/+90 region of the LOJ promoter was efficient in driving reporter gene expression in tissues like developing anthers and seeds of Arabidopsis. A positive regulation for the seed- and anther-specific expression module was contemplated within the 5′ untranslated region of the LOJ gene. However, this function was repressed in the native context by the lateral organ junction-specific expression. The present study has led to the identification of a novel lateral organ junction-specific element and an enhancer sequence in Arabidopsis with potential applications in plant genetic engineering.