Glutamate 1-semlaldehyde otra se [(S)-4-amino-5-oxopentanoate 4,5-amiomutse, EC 5.4.3.8] catalyzes the last step In the conversion of glutamate to 8-aminolevulinate of which eight molecules are needed to synthesize a chlorophyll molecule. Two fill-length cDNA clones that probably represent the homeologous Gsa genes of the two tobacco (Nicotiana tabacum) genomes have been isolated. The deduced amino acid sequences of the 468-residue-long precursor polypeptides differ by 10 amino adds. The cDNA sequence of isoenzyme 2 was inserted in reverse orientation under the control of a cauliflower mosaic virus 35S promoter derivative in an expression vector and was introduced by Agrobacteriummediated transformation into tobacco plants. Antisense (3), has been attributed to position effects arising from the random integration of antisense genes into the host chromosomes. Additionally, a lack of quantitative relationships between the steady-state level of antisense mRNA, the target RNA, and the amount of protein produced is commonly observed as well as a great variability of the antisense phenotype in response to temperature and light conditions. Cornelissen (13,14) has demonstrated with transformed tobacco cells and plants that the antisense gene can control the transcript level in the nucleus and the translation efficiency of the target mRNA in the cytoplasm independently. The target enzyme was phosphinotricine acetyltransferase produced constitutively with a cauliflower mosaic virus (CaMV) 35S promoter-driven bar gene. In a second transformation of these plants, an antisense bar gene was introduced, and the turnover of the bar mRNA in the cyto-

Arthrobacter oxydans P52 isolated from soil samples was found to degrade the phenylcarbamate herbicides phenmedipham and desmedipham cometabolically by hydrolyzing their central carbamate linkages. The phenylcarbamate hydrolase (phenmedipham hydrolase) responsible for the degradative reaction was purified to homogeneity. The enzyme was shown to be a monomer with a molecular weight of 55,000. A 41-kb wild-type plasmid (pHP52) was identified in A. oxydans P52, but not in a derivative of this strain that had spontaneously lost the ability to hydrolyze phenylcarbamates, indicating that the gene for phenylcarbamate degradation (pcd) is plasmid encoded. Determination of two partial amino acid sequences allowed the localization of the coding sequence of the pcd gene on a 3.3-kb PstI restriction fragment within pHP52 DNA by hybridization with synthetic oligonucleotides. The phenylcarbamate hydrolase was functionally expressed in Escherichia coli under control of the lacZ promoter after the 3.3-kb PstI fragment was subcloned into the vector pUC19. A stretch of 1,864 bases within the cloned Pst fragment was sequenced. Sequence analysis revealed an open reading frame of 1,479 bases containing the amino acid partial sequences determined for the purified enzyme. Sequence comparisons revealed significant homology between the pcd gene product and the amino acid sequences of esterases of eukaryotic origin. Subsequently, it was demonstrated that the esterase substrate p-nitrophenylbutyrate is hydrolyzed by phenmedipham hydrolase.

The Pem homeobox transcription factor is expressed under androgen control in the testis and epididymis. It is also transcribed in the ovary, muscle, and placenta. The mouse Pem gene promoter was cloned and sequenced. It was analyzed in transactivation tests using CV-1 and PC-3 cells expressing the AR and found to be strongly stimulated by androgens. EMSAs and mutational analysis of the Pem promoter allowed the identification of two functional androgen response elements named ARE-1 and ARE-2. They both differed from the consensus semipalindromic steroid response element and exhibited characteristics of direct repeats of the TGTTCT half-site. Unlike the steroid response element, both Pem androgen response elements were selectively responsive to androgen stimulation. Specific mutations in the left half-site of Pem ARE-1 and ARE-2, but not of the steroid response element, were still compatible with AR binding in the EMSA. In addition, Pem ARE-1, but not ARE-2 or the steroid response element, showed some flexibility with regard to spacing between half-sites. These results strongly suggest that the AR interacts differently with direct repeats than with inverted repeats, potentially leading to cis element-driven selective properties. Thus, the existence of several classes of DNA response elements might be an essential feature of differential androgen regulation.

The androgen dependency of the genes coding for the cysteine-rich secretory proteins (CRISP) was analysed in their main sites of expression. Male mice were treated with the gonadotropin-releasing hormone antagonist Ac-DNapAla-DClPhAla-DPyrAla-Ser-Tyr-DCtl-Leu-Lys(Mor)-Pro-DAla-NH 2 [DNapAla, D-2-naphthyl-Ala ; DClPhAla, D-4-chlorphenyl-Ala ; DPyrAla, D-pyridyn-3-yl-Ala ; DCtl, D-citrulline ; Lys(Mor), L-2-amino-6-(morpholin-4-yl)-hexanoic acid], and CRISP RNA levels were assessed by northern blot and competitive reverse transcriptase-mediated (RT)-PCR. In the salivary gland, CRISP-1 and to a lesser extent CRISP-3 expression was markedly reduced, in spite of an up-regulation of androgen receptor transcript levels. A down-regulation of CRISP-1 expression was also observed in the epididymis. Conversely, the levels of the testicular CRISP-2 transcripts were hardly affected at all. Female mice were ovariectomised and treated with testosterone propionate, and their salivary gland RNAs analysed. CRISP-1 and CRISP-3 RNA levels were significantly increased, and these effects were prevented by a concomitant treatment with the antiandrogen flutamide. Androgen receptor transcript levels were not affected by androgen administration but increased following antiandrogen treatment. CRISP expression during postnatal development was monitored by northern blot analysis. CRISP-1 and CRISP-2 transcripts were detected as early as 22 days after birth in the epididymis and testis, respectively, whereas CRISP-3 mRNA was visible only from day 30 in the salivary gland. A sharp increase of all CRISP levels was noted on day 40, coincident with the onset of sexual maturity. Altogether these results indicate that despite their high similarity, the CRISP genes are differentially regulated by androgens.Keywords : androgen; cysteine-rich secretory protein; epididymis; salivary gland; testis.The cysteine-rich secretory protein (CRISP) family was orig-ily of plants, which are induced upon infection by pathogens as inally described in the mouse [1, 2] and comprises evolutionarily part of the hypersensitive defense reaction [16, 17]. conserved polypeptides with a potential involvement in innate CRISP-1 is the best characterized member of the family and immunity. The CRISP genes are located on mouse chromosome its gene is mainly expressed in the corpus and cauda regions of 17 and human chromosome 6, in close vicinity to the major the epididymis, and in the male submandibular gland [1, 2, 9]. histocompatibility complex region [3Ϫ6]. They are expressed in Specific binding of its rat counterpart named acidic epididymal the specific granules of human neutrophils [7], in murine B cell glycoprotein and DE to spermatozoa heads has been reported, precursors [8], by glands with an exocrine function and by mu-leading to speculation about a role in sperm maturation [18Ϫ cosal epithelial surfaces [1, 2, 9, 10], which suggests a role in 20], but this could not be confirmed in mouse or human [9, 10, non-specific defense reactions, similar to that of defensins [11]...

Acetolactate synthase (ALS), the first enzyme in the biosynthetic pathway of leucine, valine, and isoleucine, is the biochemical target of different herbicides. To investigate the effects of repression of ALS activity through antisense gene expression we cloned an ALS gene from potato (Solanum fuberosum L. cv Désirée), constructed a chimeric antisense gene under control of the cauliflower mosaic virus 35s promoter, and created transgenic potato plants through Agrobacterium fumefaciens-mediated gene transfer. Two regenerants revealed severe growth retardation and strong phenotypical effects resembling those caused by ALS-inhibiting herbicides. Antisense gene expression decreased the steady-state leve1 of ALS mRNA in these plants and induced a corresponding decrease in ALS activity of up to 85%. This reduction was sufficient to generate plants almost inviable without amino acid supplementation. In both ALS antisense and herbicide-treated plants, we could exclude accumulation of 2-oxobutyrate and/or 2-aminobutyrate as the reason for the observed deleterious effects, but we detected elevated levels of free amino acids and imbalances in their relative proportions. Thus, antisense inhibition of ALS generated an in vivo model of herbicide action. Furthermore, expression of antisense RNA to the enzyme of interest provides a general method for validation of potential herbicide targets.