Specific association of repetitive DNA sequences with major histocompatibility genes.

The DNA sequence organization of a 17.8-kilobase segment of porcine DNA, containing a functional major histocompatibility (MHC) gene, has been studied. The DNA flanking the MHC gene contains at least 10 distinct repetitive DNA sequence elements, each of which occurs only once within the 17.8-kilobase DNA segment. Their reiteration frequencies in the genome range from 10(2) to 10(4). The genomic organization of seven of these sequence elements has been examined; all are interspersed with other, unrelated DNA sequences. These seven repeated sequences are not generally associated in the genome. However, they appear to be nonrandomly linked in MHC-associated regions of the genome: at least two additional DNA segments containing MHC-homologous DNA also contain sequences homologous to DNA fragments bearing the seven different repeats. Of the seven sequences, four can be detected in splenic total RNA. These results suggest that these repeated elements are specifically associated with the MHC locus.     

Expression of the human angiotensinogen gene in transgenic mice and transfected cells.

We have generated two lines of transgenic mice with integrated copies of a 14-kilobase pair (kb) human DNA fragment containing the angiotensinogen gene, which includes 1.3 kb of 5'- and 3'-flanking regions. In both transgenic lines, a considerable quantity of the correctly initiated and processed angiotensinogen mRNA was detected in the liver and it was detectable in heart. Unexpectedly, mRNA for the transgene was accumulated in the kidney, where is normally the minor source of angiotensinogen, to levels comparable to that in the liver. In addition, an in vitro transfection analysis suggested that the 1.3-kb 5'-flanking sequences are essential for expression of the angiotensinogen gene in hepatic and renal cells and that neither DNA segment within the 14-kb construct contributes significantly to repression of the gene expression in renal cells.     

Expression of a class I MHC transgene: regulation by a tissue-specific negative regulatory DNA sequence element

In vivo patterns of expression of a miniature swine class I major histocompatibility gene, PD7, were analyzed both in situ in the pig, and in transgenic mice. Structural analysis of PD7 DNA sequences revealed that PD7 is highly homologous to the pig gene PD1, which encodes a classical transplantation antigen. Despite the extensive homology, PD7 is expressed in situ at markedly lower levels than PD1 in nearly all tissues. Introduction of PD7 into mice results in a pattern of PD7 expression in the transgenic animals that parallels that observed in situ in the pig. Comparison of two lines of PD7 transgenic mice, which differ only in the extent of 5' flanking sequence, reveals the presence of a silencer element. The silencer activity is tissue specific: differences in PD7 expression are observed only in lymphoid tissues and skin. Skin from both lines of transgenics mediates graft rejection, but the rate of rejection correlates with the level of PD7 expression.     

Gene transfer using recombinant rabbit hemorrhagic disease virus capsids with genetically modified DNA encapsidation capacity by addition of packaging sequences from the L1 or L2 protein of human papillomavirus type 16

The aim of this study was to produce gene transfer vectors consisting of plasmid DNA packaged into virus-like particles (VLPs) with different cell tropisms. For this purpose, we have fused the N-terminally truncated VP60 capsid protein of the rabbit hemorrhagic disease virus (RHDV) with sequences which are expected to be sufficient to confer DNA packaging and gene transfer properties to the chimeric VLPs. Each of the two putative DNA-binding sequences of major L1 and minor L2 capsid proteins of human papillomavirus type 16 (HPV-16) were fused at the N terminus of the truncated VP60 protein. The two recombinant chimeric proteins expressed in insect cells self-assembled into VLPs similar in size and appearance to authentic RHDV virions. The chimeric proteins had acquired the ability to bind DNA. The two chimeric VLPs were therefore able to package plasmid DNA. However, only the chimeric VLPs containing the DNA packaging signal of the L1 protein were able efficiently to transfer genes into Cos-7 cells at a rate similar to that observed with papillomavirus L1 VLPs. It was possible to transfect only a very limited number of RK13 rabbit cells with the chimeric RHDV capsids containing the L2-binding sequence. The chimeric RHDV capsids containing the L1-binding sequence transfer genes into rabbit and hare cells at a higher rate than do HPV-16 L1 VLPs. However, no gene transfer was observed in human cell lines. The findings of this study demonstrate that the insertion of a DNA packaging sequence into a VLP which is not able to encapsidate DNA transforms this capsid into an artificial virus that could be used as a gene transfer vector. This possibility opens the way to designing new vectors with different cell tropisms by inserting such DNA packaging sequences into the major capsid proteins of other viruses.     

Cloning the Gene for the Malolactic Fermentation of Wine from Lactobacillus delbrueckii in Escherichia coli and Yeasts

The gene responsible for the malolactic fermentation of wine was cloned from the bacterium Lactobacillus delbrueckii into Escherichia coli and the yeast Saccharomyces cerevisiae. This gene codes for the malolactic enzyme which catalyzes the conversion of l-malate to l-lactate. A genetically engineered yeast strain with this enzymatic capability would be of considerable value to winemakers. L. delbrueckii DNA was cloned in E. coli on the plasmid pBR322, and two E. coll clones able to convert l-malate to l-lactate were selected. Both clones contained the same 5-kilobase segment of L. delbrueckii DNA. The DNA segment was transferred to E. coli-yeast shuttle vectors, and gene expression was analyzed in both hosts by using enzymatic assays for l-lactate and l-malate. When grown nonaerobically for 5 days, E. coli cells harboring the malolactic gene converted about 10% of the l-malate in the medium to l-lactate. The best expression in S. cerevisiae was attained by transfer of the gene to a shuttle vector containing both a yeast 2-mum plasmid and yeast chromosomal origin of DNA replication. When yeast cells harboring this plasmid were grown nonaerobically for 5 days, ca. 1.0% of the l-malate present in the medium was converted to l-lactate. The L. delbrueckii controls grown under these same conditions converted about 25%. A laboratory yeast strain containing the cloned malolactic gene was used to make wine in a trial fermentation, and about 1.5% of the l-malate in the grape must was converted to l-lactate. Increased expression of the malolactic gene in wine yeast will be required for its use in winemaking. This will require an increased understanding of the factors governing the expression of this gene in yeasts.     

Level of expression of the tomato rbcS-3A gene is modulated by a far upstream promoter element in a developmentally regulated manner.

By Agrobacterium-mediated transformation we have demonstrated that a 1.10-kilobase promoter sequence from the tomato rbcS-3A gene confers light-inducible and organ-specific expression upon fusion to the bacterial chloramphenicol acetyltransferase gene. A biphasic expression profile was obtained by 5' deletion analysis of this promoter, indicating the presence of both positive and negative regulatory elements. A severe reduction in the level of expression was observed when the 5'-terminal 90 base pairs were deleted from the 1.10-kilobase promoter. DNA sequence elements responsible for light inducibility and organ specificity of the gene reside within the -374 base pairs of the proximal part of the promoter and the sequences spanning from -374 to -205 are essential for promoter function. The DNA sequences upstream from -374 modulate the level of expression in leaf tissue; this modulation is under developmental control.     

Spontaneous deletion of a 20-kilobase DNA segment carrying genes specifying isopropylbenzene metabolism in Pseudomonas putida RE204.

The genes encoding isopropylbenzene metabolism in Pseudomonas putida RE204 are readily lost in two ways: by loss (curing) of plasmid pRE4 which specifies the catabolic pathway and by deletion from pRE4 of an approximately 20-kilobase segment of DNA carrying the catabolic genes. The presence of DNA sequences at the ends of the catabolic gene region sharing homology with one another suggests that the deletions result from recombination events between these homologous sequences.