Gene transfer and expression in mouse preimplantation embryos by recombinant adenovirus vector

Replication-defective recombinant adenovirus, Adex4SRLacZL, was used as a vector for transferring exogenous genes in mouse zona pellucida-free eggs at the pronuclear stage. The vector contained the E. coli LacZ reporter gene under the control of the SRα promoter (SV40 early promoter-fused HTLV-I LTR), and the expression of the reporter gene was examined during preimplantation development in culture. Histochemical staining of the embryos for β-galactosidase activity showed that the exogenous LacZ gene as expressed in 98% of the embryos at the morula-blastocyst stages. As in the microinjection method, the exogenous genes could be pursued from the 2-cell stage. Neither apparent morphological changes nor cytotoxic effects were observed. Both the percentages of embryos expressing reporter genes and the rate of development to the blastocyst stage were higher in the adenovirus vector-treated embryos than in the microinjected ones. These results suggest that the adenovirus vector system is a useful tool in investigating the genetic control of early mammalian development. © 1995 wiley-Liss, Inc.     

Structural organization and differential expression of three stilbene synthase genes located on a 13 kb grapevine DNA fragment

A 13 kb DNA fragment was isolated from a grapevine (Vitis var. Optima) genomic library by hybridizing with elicitor-induced stilbene synthase cDNA as a probe. After fragmentation with Eco RI, subcloning and sequencing, two full-size stilbene synthase genes (Vst1 and Vst2) and the 3 end of a third stilbene synthase gene (Vst3) were located within the 13 kb fragment. Vst1 and Vst2, differing only slightly in the coding region, are distinguished in the intron size and in the structure of the promoter region. The 5 flanking region of gene Vst1 contains a TATAA box at nucleotide –48. The substantial structural differences found for the promoters of the two genes are paralleled by a striking difference in the expression of the two genes in elicitor-treated cells. Moreover, the accumulation upon elicitation of six different stilbene synthase mRNAs was studied and found to differ by two orders of magnitude.     

Over-production of the D1 protein of photosystem II reaction centre in the cyanobacterium Synechococcus sp. PCC 7942

The unicellular cyanobacterium Synechococcus sp. PCC 7942 has three psbA genes encoding two different forms of the photosystem II reaction centre protein D1 (D1:1 and D1:2). The level of expression of these psbA genes and the synthesis of D1:1 and D1:2 are strongly regulated under varying light conditions. In order to better understand the regulatory mechanisms underlying these processes, we have constructed a strain of Synechococcus sp. PCC 7942 capable of over-producing psbA mRNA and D1 protein. In this study, we describe the over-expression of D1:1 using a tac-hybrid promoter in front of the psbAI gene in combination with lacI ^Q repressor system. Over-production of D1:1 was induced by growing cells for 12 h at 50 mol photons m^-2 s^-1 in the presence of 40 or 80 g/ml IPTG. The amount of psbAI mRNA and that of D1:1 protein in cells grown with IPTG was three times and two times higher, respectively. A higher concentration of IPTG (i.e., 150 g/ml) did not further increase the production of the psbAI message or D1:1. The over-production of D1:1 caused a decrease in the level of D1:2 synthesised, resulting in most PSII reaction centres containing D1:1. However, the over-production of D1:1 had no effect on the pigment composition (chlorophyll a or phycocyanin/number of cells) or the light-saturated rate of photosynthesis. This and the fact that the total amounts of D1 and D2 proteins were not affected by IPTG suggest that the number of PSII centres within the membranes remained unchanged. From these results, we conclude that expression of psbAI can be regulated by using the tac promoter and lacI ^Q system. However, the accumulation of D1:1 protein into the membrane is regulated by the number of PSII centres.     

Meristem-specific gene expression directed by the promoter of the S-phase-specific gene,cyc07, in transgenic Arabidopsis

A genomic clone for the cyc07 gene, which is expressed specifically at the S phase during the cell cycle in synchronous cultures of periwinkle (Catharanthus roseus) cells, was isolated. Determination of the nucleotide sequence of the clone revealed that the cyc07 gene consists of seven exons separated by six introns. Genomic Southern analysis indicated that the cyc07 gene is present as a single copy per haploid genome in periwinkle. Expression of related genes was detected in a wide range of other plants. Transgenic Arabidopsis plants were generated that expressed the gene for -glucuronidase (GUS) under the control of the promoter of the cyc07 gene. The tissue-specific pattern of expression directed by the promoter was investigated by analysis of GUS activity. Histochemical tests demonstrated that 589 bp of the 5-upstream sequence of the cyc07 gene could direct specifical expression of the GUS reporter gene in meristematic tissues in transgenic plants. The spatial pattern of expression directed by the promoter was closely correlated with meristematic activity and cell proliferation, suggesting an association between the function of the cyc07 gene and cell proliferation.     

An autonomously propagating luciferase-encoding vector for Dictyostelium discoideum

We have constructed a luc reporter vector for Dictyostelium discoideum using a 626-bp fragment from the nuclear-associated plasmid Ddp2. The ori from Ddp2 is localized within this fragment and was used to provide an autonomous replication sequence for the reporter vector. This reporter vector was stably retained in D. discoideum AX3K cells without alteration. The vector molecule was also found to exist in relatively low copy number compared to other Dictyostelium vectors in the transformed cells. We demonstrated the utility of this vector as a reporter vector with glycogen synthase promoter/luc fusions of varying sizes.     

Conformational properties of the —35 region of the trp promoter in solution: comparison of the wild-type sequence with an AT transversion

The majority of the ¹H NMR resonances of the protons in a tetradecamer containing the —35 region of the trp promoter d(GCTGTTGACAATTA): d(TAATTGTCAACAGC) and in the TA transversion have been assigned. The conformational properties of the nucleotides have been determined and compared in the two duplexes. Analysis of spin-spin coupling and NOES shows that all sugar puckers are in the south domain (i.e. near C2 endo) and the glycosidic torsion angles are anti (110°). The NMR data are consistent with the duplex being in the B family of conformations. Significant differences in chemical shifts between the two molecules were observed only for nearest neighbours to the transversion site, suggesting the absence of long range conformational effects. This was confirmed by the similarity of coupling constants and NOEs. Other properties are also not greatly affected at positions more than two base pairs from the mutation site. These results are consistent with the hypothesis that unconstrained oligonucleotides are highly flexible, and can readily accommodate significant perturbations of the local structure, such as a transversion.