Expression of endogenous and microinjected hsp 30 genes in early Xenopus laevis embryos

In the present study, we have examined the regulation of expression of a newly isolated member of the hsp 30 gene family, hsp 30C. Using RT-PCR, we found that this gene was first heat-inducible at the tailbud stage of development. We also examined the expression of two microinjected modified hsp 30C gene constructs in Xenopus embryos. One of the constructs had 404 bp of hsp 30C 5′-flanking region, whereas the other had 3.6 kb. Both gene constructs had 1 kb of 3′-flanking region. RT-PCR assays were employed to detect the expression of these microinjected genes. The presence of extensive 5′- and 3′-flanking regions of the hsp 30C gene did not confer proper developmental regulation, since heat-inducible expression of both of the microinjected constructs was detectable at the midblastula stage. The premature expression of the microinjected hsp 30 gene was not a result of high plasmid copy number or the presence of plasmid DNA sequences. These results suggest that the microinjected genes contain all the cis-acting DNA sequences required for correct heat-inducible regulation but do not contain the elements required for the proper regulation of hsp 30 gene expression during development. It is possible that regulatory elements controlling the developmental expression of the hsp30 genes may reside upstream or downstream of the entire cluster. © 1993Wiley-Liss, Inc.     

Growth-responsive expression from the murine thymidine kinase promoter: genetic analysis of DNA sequences

As a first step toward elucidating the biochemical basis of gene regulation at the G1-S boundary of the cell cycle, we have identified regions of the murine thymidine kinase (TK) promoter sufficient to confer appropriately growth-responsive expression to a heterologous gene. Using a series of TK promoter-chloramphenicol acetyltransferase (CAT) gene fusion constructs, we have identified sequences located between -174 base pairs upstream and +159 base pairs downstream of the TK translation initiation site that are sufficient to drive efficient S phase-specific expression of the CAT reporter gene in transfected murine fibroblasts. Both deletion analysis and site-specific mutagenesis experiments indicated that an Sp1 consensus binding site is critical to the activity of this promoter. Synchronized populations of BALB/c 3T3 cells stably transfected with either TK promoter-CAT fusion constructs or TK promoter-beta-globin fusion constructs expressed their respective reporter genes in an S phase-specific manner following serum stimulation. In each case, reporter gene expression was reduced during quiescence and G1 and rose upon entry of cells into S phase. The TK sequences included in these constructs therefore contained information sufficient to confer S phase-specific regulation to these two reporter genes. These results set the stage for a more detailed analysis of the sequences and trans-acting factors responsible for regulating murine TK gene expression and may lead to insights into the control of proliferation in normal and transformed cells.     

Regulation of the cell expansion gene RHD3 during Arabidopsis development

The RHD3 (ROOT HAIR DEFECTIVE3) gene encodes a putative GTP-binding protein required for appropriate cell enlargement in Arabidopsis. To obtain insight into the mechanisms of RHD3 regulation, we conducted a molecular genetic dissection of RHD3 gene expression and function. Gene fusion and complementation studies show that the RHD3 gene is highly expressed throughout Arabidopsis development and is controlled by two major regulatory regions. One regulatory region is located between -1,500 and -600 bp upstream of the RHD3 gene and is required for vascular tissue expression. The other region is intragenically located and includes the 558-bp first intron, which is responsible for high-level expression of RHD3 throughout the plant. The presence and location of this intron is essential for gene function because constructs lacking this intron or constructs with the intron in an abnormal position are unable to functionally complement the rhd3 mutations. We also analyzed the role of other RHD genes and the plant hormones auxin and ethylene in RHD3 regulation, and we determined that these act downstream or independently from the RHD3 pathway. This study shows that multiple levels of regulation are employed to ensure the appropriate expression of RHD3 throughout Arabidopsis development.     

Regulation of tomato leaf curl viral gene expression in host tissues

The regulation of expression of the two virion-sense (V1 and V2) and four complementary-sense (C1, C2, C3, and C4) open reading frames (ORFs) of Tomato leaf curl virus (TLCV) was studied in both stably and transiently transformed Nicotiana tabacum tissues with fusions with the beta-glucuronidase (GUS) reporter gene. GUS-expressing transgenic lines were obtained with each of the four complementary-sense gene-GUS fusion constructs and with truncated versions of the virion-sense gene-GUS fusion constructs (V1GUSdeltaC and V2GUSdeltaC) lacking complementary-sense sequences encoding the C1, C2, and C3 ORFs. However, little or no GUS expression was observed in kanamycin-resistant plants transformed with full-length, virion-sense gene constructs (V1GUS and V2GUS) constituting the complete viral genome. In contrast, V1GUS and V2GUS were found to direct high-level GUS expression in transient assays with tobacco protoplasts, suggesting that integration of viral constructs containing functional, complementary-sense genes may lead to repression or deletion of the introduced constructs in transgenic tissues. V2GUS expression in the transient protoplast assay was found to be severely curtailed by specific mutation of the C2 ORF, supporting a role for the C2 protein in transactivation of TLCV virion-sense gene expression. TLCV ORF-GUS constructs displayed distinctive tissue expression patterns in transgenic tobacco plants that could be divided into constitutive (C1, C4, and V2GUSdeltaC), predominantly vascular (C2, C3), or reduced expression in cells associated with the vascular bundles (V1GUSdeltaC). The significance of these results is discussed in terms of current models of gene function and regulation in geminiviruses.     

Position independence and proper developmental control of gamma-globin gene expression require both a 5'' locus control region and a downstream sequence element.

We have analyzed the expression of human gamma-globin genes during development in F2 progeny of transgenic mice carrying two types of constructs. In the first type, gamma-globin genes were linked individually to large (approximately 4-kb) sequence fragments spanning locus control region (LCR) hypersensitive site 2 (HS2) or HS3. These LCR fragments contained not only the core HS elements but also extensive evolutionarily conserved flanking sequences. The second type of construct contained tandem gamma- and beta-globin genes linked to identical HS2 or HS3 fragments. We show that gamma-globin expression in transgenic mice carrying HS2 gamma or HS3 gamma constructs is highly sensitive to position effects and that such effects override the cis regulatory elements present in these constructs to produce markedly different developmental patterns of gamma-globin expression in lines carrying the same transgene. In contrast, gamma-globin expression in both HS2 gamma beta and HS3 gamma beta mice is sheltered from position effects and the developmental patterns of gamma-globin expression in lines carrying the same transgene are identical and display stage-specific regulation. The results suggest that cis regulatory sequences required for proper developmental control of fetal globin expression in the presence of an LCR element reside downstream from the gamma genes.     

PPARα and PPARγ regulate the nucleoside transporter hENT1

Human equilibrative nucleoside transporter 1 (hENT1) is an important determinant for nucleoside analog based chemotherapy success. Preliminary data suggest hENT1 regulation by PPARs. Using A2780 cells, we investigated the role of PPARs on hENT1 expression and activity. PPARα and PPARγ agonists, Wy14,643 and RGZ, increased hENT1 expression, but only PPARα activation or overexpression resulted in higher hENT1 transport activity. On the other hand, promoter analysis showed two putative PPRE in hENT1 promoter and luciferase-coupled promoter constructs were generated and analyzed. Our results suggest that PPARα-but not PPARγ-mediated expression regulation of hENT1 is PPRE-dependent. In conclusion, PPARα and PPARγ activation modulate hENT1 expression.     

Differential expression of the human gonadotropin alpha gene in ectopic and eutopic cells.

We have analyzed the regulation of the alpha gonadotropin gene in eutopic placental cells and ectopic tumor cells by constructing a series of plasmid vectors containing alpha genomic 5' flanking DNA placed upstream of the gene encoding the bacterial enzyme chloramphenicol acetyltransferase (CAT). These plasmid DNAs were transfected into a eutopic (JAr) and an ectopic (HeLa) cell line. Both cell types expressed the CAT gene from plasmid constructs containing as much as 1,500 base pairs (bp) and as little as 140 bp of alpha 5' flanking DNA; JAr cells were considerably more efficient than HeLa cells. Ectopic and eutopic cells differed qualitatively in their expression from these alpha-CAT constructs when cells were treated with cAMP or butyrate. Butyrate induced alpha expression in HeLa cells but not in JAr cells, while cAMP induced expression in JAr cells. These results are consistent with and extend previous observations suggesting that there are cell-specific differences in the regulation of alpha gene expression in ectopic and eutopic cells. However, by using deletion constructs of the alpha-CAT gene, we found that the basal expression and cell-specific induction of the alpha gene in ectopic and eutopic cells were dependent on the same 140 bp of alpha 5' flanking DNA. These 140 bp were sequenced and found to contain a 9-bp stretch of DNA homologous with the consensus viral enhancer sequence. Such features of alpha expression common to both ectopic and eutopic cells may be involved in the coordinate expression of the alpha gene and the tumorigenic phenotype observed in each cell type.     

Parallel screening and optimization of protein constructs for structural studies

A major challenge in structural biology remains the identification of protein constructs amenable to structural characterization. Here, we present a simple method for parallel expression, labeling, and purification of protein constructs (up to 80 kDa) combined with rapid evaluation by NMR spectroscopy. Our approach, which is equally applicable for manual or automated implementation, offers an efficient way to identify and optimize protein constructs for NMR or X‐ray crystallographic investigations.     

Optimized bacterial expression and purification of the c-Src catalytic domain for solution NMR studies

Progression of a host of human cancers is associated with elevated levels of expression and catalytic activity of the Src family of tyrosine kinases (SFKs), making them key therapeutic targets. Even with the availability of multiple crystal structures of active and inactive forms of the SFK catalytic domain (CD), a complete understanding of its catalytic regulation is unavailable. Also unavailable are atomic or near-atomic resolution information about their interactions, often weak or transient, with regulating phosphatases and downstream targets. Solution NMR, the biophysical method best suited to tackle this problem, was previously hindered by difficulties in bacterial expression and purification of sufficient quantities of soluble, properly folded protein for economically viable labeling with NMR-active isotopes. Through a choice of optimal constructs, co-expression with chaperones and optimization of the purification protocol, we have achieved the ability to bacterially produce large quantities of the isotopically-labeled CD of c-Src, the prototypical SFK, and of its activating Tyr-phosphorylated form. All constructs produce excellent spectra allowing solution NMR studies of this family in an efficient manner. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Light-regulated expression of the pea plastocyanin gene is mediated by elements within the transcribed region of the gene

Expression of the pea plastocyanin gene ( PetE ) is regulated by light in both pea and transgenic tobacco plants. However, the PetE promoter with the 5' untranslated leader region does not direct light-regulated expression of the GUS reporter gene in transgenic tobacco. This suggested that sequences downstream of the translation start of the PetE gene are required for light-regulated expression. To investigate this possibility the expression of a series of chimeric gene constructs in transgenic tobacco plants was examined to assess the contributions of the promoter, the 5' untranslated leader region, the coding region and the 3' region of the PetE gene to light-regulated expression. Both the coding region and the 5' untranslated leader region of the PetE gene were found to be required for full light regulation. Full light regulation of chimeric gene constructs containing the cauliflower mosaic virus (CaMV) 35S promoter required the deletion of CaMV 5' leader and polylinker sequences from the constructs. The presence of CaMV and polylinker sequences at the 5' end of the PetE leader masked the light regulation directed by the transcribed region of the pea PetE gene.     

A synergistic effect of suppressive CGG codon in +2 position and downstream CAT repeats for efficient heterologous protein expression in Escherichia coli

The negative effect of NGG codons at +2 position has been well documented for the down regulation of recombinant protein expression in Escherichia coli. But this is not true when certain specific sequences are present in the downstream of NGG codons. This has been proved in our study while expressing human Erythropoietin (EPO) in E. coli GJ1158. Towards this, nine recombinant constructs were made and their expression profile was compared. In our results, we found that the suppressive nature of NGG codon (GGG, CGG) in the +2 position was overcome by imposing a downstream CAT repeat motif. The expression of EPO levels is higher in the constructs having the combination of both CGG codon at 2nd position and CAT repeats than the other constructs having either CGG or CAT repeat alone. In addition, it is also interesting to note that increasing number of CAT repeats shows increased expression levels.     

Efficient Transfection Strategy for the Spatiotemporal Control of Gene Expression in Zebrafish

Functional analyses of gene function by knockdown and expression approaches strongly enhance the genetic study of development. In vivo application of the introduction of inhibitors of gene expression, mRNA, and expression constructs in the target region make it possible to perform region- and stage-specific regulation of gene function in a simple manner. As a basic tool for the conditional regulation of gene expression in target tissue, we present methods for the efficient introduction of antisense morpholino oligonucleotide (MO), mRNA, and expression plasmid constructs into early and later stage zebrafish embryo and larva. Lipofection of a neuron-specific expression construct plasmid encoding green fluorescent protein (GFP) into optic vesicle resulted in clear GFP expression in the retinotectal pathway in hatched larva. Co-lipofection of MO and GFP mRNA to the presumptive head region resulted in brain-specific knockdown of the gene in mid-stage embryos.     

Expression of soybean lectin gene deletions in tobacco

A series of constructs containing the developmentally regulated soybean lectin gene (Le1) were used to transform tobacco plants in order to assess developmental and quantitative regulation conferred by flanking sequences. The largest of the lectin constructs contained approximately 3,000 base pairs (bp) of Le1 5 flanking region and 1,500 bp of the 3 flanking region. The smallest construct contained no 5 flanking region and 194 bp of the 3 flanking region. ELISA assays of lectin in individual tobacco seeds and Southern blot analyses confirmed that most constructs were inherited as unique insertion events. Maximal expression of Le1 required more than 338 bp of 5 sequence, indicating that far upstream factors are involved in quantitative control of lectin expression. Lectin expression declined more than 80% between deletions with 1,700 versus 338 bp of 5 flanking sequence. In contrast, developmental control of lectin expression was maintained by Le1 inserts with only 190 bp of 5 sequence. The lectin promoter offers a potential means to target high levels of gene expression to the developing seeds of soybean or other dicotyledonous plants.