A deletion adjacent to the maize transposable element Mu-1 accompanies loss of Adh1 expression.

Insertion of the maize transposable element Mu-1 into the first intron of the alcohol dehydrogenase locus (Adh1) of maize produced mutant Adh1-S3034 with 40% of the wild-type level of protein and mRNA. Continued instability at this locus resulted in secondary mutations with lower levels of protein expression. One of these, Adh1-S3034a, has no detectable ADH1 expression. This paper describes the precise nature of the changes in the Adh1 gene that gave rise to the S3034a allele. The Mu-1 element is still present in the mutant, but Adh1 sequences immediately adjacent to the element are deleted. The deletion starts precisely at the Mu-1 insertion site and extends 74 bp leftward removing part of the first intron, the intron:exon junction and 2 bp of the eleventh amino acid codon in the first exon of the gene. Tests for reversion within the somatic tissue of plants show that mutant S3034a, unlike its progenitor, is stably null for ADH1 activity.     

Isolation and characterization of the genomic region from Drosophila kuntzei containing the Adh and Adhr genes

The nucleotide sequences of the Adh and Adhr genes of Drosophila kuntzei were derived from combined overlapping sequences of clones isolated from a genomic library and from cloned PCR and inverse-PCR fragments. Only a proximal promoter was detected upstream of the Adh gene, indicating that D. kuntzei Adh is regulated by a one-promoter system. Further upstream of the Adh structural gene, an adult enhancer region (AAE) was found that contains most of the regulatory sequences described for AAEs of other Drosophila species. Analysis of the ADH protein showed an amino acid change from valine to threonine in the active site at position 189 which is also found in D. funebris but is otherwise unique among DROSOPHILA: This difference alone may be responsible for the very low ADH activity found in this species and may cause a difference in substrate usage pattern. Codon bias in Adh and Adhr was comparable and found to be very low compared with other species. Phylogenetic analysis showed that D. kuntzei is closest related to D. funebris and D. immigrans. The time of divergence between D. kuntzei and D. funebris was estimated to be 14.2-20.2 Myr and that between D. kuntzei-D. funebris and D. immigrans to be 30.8-44.0 Myr. An analysis of the genetic variation in the Adh gene and upstream sequences of four European strains showed that this gene was highly variable. Overall nucleotide diversity (pi) was 0.0139, which is two times higher than that in D. melanogaster.     

Correct developmental expression of a cloned alcohol dehydrogenase gene transduced into the Drosophila germ line

We have used P-element-mediated transformation to introduce a cloned Drosophila alcohol dehydrogenase (Adh) gene into the germ line of ADH null flies. Six independent transformants expressing ADH were identified by their acquired resistance to ethanol. Each transformant carries a single copy of the cloned Adh gene in a different chromosomal location. Four of the six transformant lines exhibit normal Adh expression by the following criteria: quantitative levels of ADH enzyme activity in larvae and adults; qualitative tissue specificity; the size of stable Adh mRNA; and the characteristic developmental switch in utilization of two different Adh promoters. The remaining two transformants express ADH enzyme activity with the correct tissue specificity, but at a lower level than wild type. These results demonstrate that an 11.8 kb chromosomal fragment containing the Adh gene includes the cis-acting sequences necessary for its correct developmental expression, and that a variety of chromosomal sites permit proper Adh gene function.     

ADH enzyme activity and Adh gene expression in Drosophila melanogaster lines differentially selected for increased alcohol tolerance

In Drosophila melanogaster, alcohol dehydrogenase (ADH) activity is essential for ethanol tolerance, but its role may not be restricted to alcohol metabolism alone. Here we describe ADH activity and Adh expression level upon selection for increased alcohol tolerance in different life-stages of D. melanogaster lines with two distinct Adh genotypes: Adh(FF) and Adh(SS). We demonstrate a positive within genotype response for increased alcohol tolerance. Life-stage dependent selection was observed in larvae only. A slight constitutive increase in adult ADH activity for all selection regimes and genotypes was observed, that was not paralleled by Adh expression. Larval Adh expression showed a constitutive increase, that was not reflected in ADH activity. Upon exposure to environmental ethanol, sex, selection regime life stage and genotype appear to have differential effects. Increased ADH activity accompanies increased ethanol tolerance in D. melanogaster but this increase is not paralleled by expression of the Adh gene.     

Transposon-mediated mutations in the untranslated leader of maize Adh1 that increase and decrease pollen-specific gene expression.

The unstable mutation Adh1-Fm335 contains a Dissociation (Ds1) transposable element at position +53 in the untranslated leader of the maize Alcohol dehydrogenase-1 (Adh1) gene. Excision of Ds1 is known to generate new alleles with small additions and rearrangements of Adh1 DNA. We characterized 16 revertant alleles with respect to ADH1 activity levels in scutellum (nutritive tissue of the seed), anaerobic root, and pollen. Whereas gene expression was not different from the wild type in the sporophytic tissues of the scutellum and anaerobic root, there were strong allelic differences in pollen. One allele underexpressed pollen ADH1 at 48% of the wild-type level, and another overexpressed pollen ADH1 at 163% of the wild-type level. Quantitative RNase protection assays demonstrated that the mutant phenotypes reflected changes in the levels of steady state mRNA in pollen. These data provide a definitive demonstration of an overexpression mutant in plants and further show that marked increases in mRNA levels can follow minor alterations in central untranslated leader sequences. The nucleotide sequence of 12 new revertant alleles and the molecular mechanisms responsible for pollen-specific gene expression are discussed.     

Functional Analysis of Two Matrix Attachment Region (MAR) Elements in Transgenic Maize Plants

Matrix attachment regions (MARs) are binding sites for nuclear scaffold proteins in vitro, and are proposed to mediate the attachment of chromatin to the nuclear scaffold in vivo. Previous reports suggest that MAR elements may stabilize transgene expression. Here, we tested the effects of two maize MAR elements (P-MAR from the P1-rr gene, and Adh1-MAR from the adh1 gene) on the expression of a gusA reporter gene driven by three different promoters: the maize p1 gene promoter, a wheat peroxidase (WP) gene promoter, or a synthetic promoter (Rsyn7). The inclusion of P-MAR or Adh1-MAR on P::GUS transgene constructs did not reduce variation in the levels of GUS activity among independent transformation events, nor among the progeny derived from each event. The Adh1-MAR element did not affect GUS expression driven by the WP promoter, but did modify the spatial pattern of expression of the Rsyn7::GUS transgene. These results indicate that, in transgenic maize plants, the effects of MAR elements can vary significantly depending upon the promoter used to drive the transgene.     

Deletion of a conserved regulatory element in the Drosophila Adh gene leads to increased alcohol dehydrogenase activity but also delays development

In vivo levels of enzymatic activity may be increased through either structural or regulatory changes. Here we use Drosophila melanogaster alcohol dehydrogenase (ADH) in an experimental test for selective differences between these two mechanisms. The well-known ADH-Slow (S)/Fast (F) amino acid replacement leads to a twofold increase in activity by increasing the catalytic efficiency of the enzyme. Disruption of a highly conserved, negative regulatory element in the Adh 3' UTR also leads to a twofold increase in activity, although this is achieved by increasing in vivo Adh mRNA and protein concentrations. These two changes appear to be under different types of selection, with positive selection favoring the amino acid replacement and purifying selection maintaining the 3' UTR sequence. Using transgenic experiments we show that deletion of the conserved 3' UTR element increases adult and larval Adh expression in both the ADH-F and ADH-S genetic backgrounds. However, the 3' UTR deletion also leads to a significant increase in developmental time in both backgrounds. ADH allozyme type has no detectable effect on development. These results demonstrate a negative fitness effect associated with Adh overexpression. This provides a mechanism whereby natural selection can discriminate between alternative pathways of increasing enzymatic activity.     

Induction and repression of the Drosophila Sgs-3 glue gene are mediated by distinct sequences in the proximal promoter.

The normal developmental expression of the Drosophila salivary gland secretion protein gene Sgs-3 requires the interaction of a distal and proximal regulatory element. A deletion/replacement analysis of the proximal promoter in stably transformed lines shows that induction of an Sgs-3/Adh fusion gene is normal if sequences from +10 to -50 are replaced by those of the hsp70 gene. Sequences between -98 and -50 are necessary for this expression but there is internal redundancy within this region as two distinct upstream sequences of 18 and 22 bp respectively are sufficient for stage- and tissue-specific expression, albeit at reduced levels. A point mutation at -53 eliminates the ecdysone-mediated repression of the Sgs-3 promoter at pupariation. We report mosaicisms of expression within the salivary gland for a number of stably transformed lines.