The Cre protein encoded by the coliphage P1 is a 38-kDa protein that efficiently promotes both intra-and intermolecular synapsis and recombination of DNA both in Escherichia coli and in vitro. Recombination occurs at a specific site, called lox, and does not require any other protein factors. The Cre protein is shown here also to be able to cause synapsis of DNA and site-specific recombination in a mammalian cell line. A stable mouse cell line was established that expresses the Cre protein under the control of the Cd2+-inducible metallothionein I gene promoter. DNA recombination was monitored with DNA substrates containing two directly repeated lox sites. One such substrate is a circular plasmid with two directly repeated lox sites (lox2) flanking a marker gene and was introduced into cells by Ca3(P04)2 transformation. As a second substrate we used a pseudorabies virus (a herpesvirus) containing a lox2 insertion designed to provide a sensitive detection system for recombination. In both cases, site-specific recombination in vivo is dependent on the presence of the Cre protein and occurs specifically at the 34-base-pair lox sites. These results demonstrate the controlled site-specific synapsis ofDNA and recombination by a prokaryotic protein in mammalian cells and suggest that Cre-mediated site-specific recombination may be a useful tool for understanding and modulating genome rearrangements in eukaryotes.

The mouse aspartyl ␤-hydroxylase gene (Asph, BAH) has been cloned and characterized. The mouse BAH gene spans 200 kilobase pairs of genomic DNA and contains 24 exons. Of three major BAH-related transcripts, the two largest (6,629 and 4,419 base pairs) encode fulllength protein and differ only in the use of alternative polyadenylation signals. The smallest BAH-related transcript (2,789 base pairs) uses an alternative 3 terminal exon, resulting in a protein lacking a catalytic domain. Evolutionary conservation of this noncatalytic isoform of BAH (humbug) is demonstrated in mouse, man, and Drosophila. Monoclonal antibody reagents were generated, epitope-mapped, and used to definitively correlate RNA bands on Northern blots with protein species on Western blots. The gene for mouse junctin, a calsequestrin-binding protein, was cloned and characterized and shown to be encoded from the same locus. When expressed in heart tissue, BAH/humbug preferably use the first exon and often the fourth exon of junctin while preserving the reading frame. Thus, three individual genes share common exons and open reading frames and use separate promoters to achieve differential expression, splicing, and function in a variety of tissues. This unusual form of exon sharing suggests that the functions of junctin, BAH, and humbug may be linked.

The cre gene of coliphage P1 encodes a 38 kDa protein which efficiently promotes both intra- and intermolecular recombination at specific 34 bp sites called loxP. To demonstrate that the Cre protein can promote DNA recombination at loxP sites resident on a mammalian chromosome, a mouse cell line was constructed containing two directly repeated loxP sites flanking a 2.5 kb yeast DNA fragment and inserted between the SV40 promoter and the neo structural gene to disrupt expression of the neo gene. Expression of the cre gene in this cell line results in excision of the intervening yeast DNA and thus permits sufficient expression of the neo gene to allow cell growth in high concentrations of G418. Southern analysis indicated that Cre-mediated excision occurred at the loxP sites. In the absence of the cre gene such excisive events are quite rare. Cre-mediated recombination should thus be quite useful in effecting a variety of genomic rearrangements in eukaryotic cells.

The BAH genomic locus encodes three distinct proteins: junctin, humbug, and BAH. All three proteins share common exons, but differ significantly based upon the use of alternative terminal exons. The biological roles of BAH and humbug and their functional relationship to junctin remain unclear. To evaluate the role of BAH in vivo, the catalytic domain of BAH was specifically targeted such that the coding regions of junctin and humbug remained undisturbed. BAH null mice lack measurable BAH protein in several tissues, lack aspartyl ␤-hydroxylase activity in liver preparations, and exhibit no hydroxylation of the epidermal growth factor (EGF) domain of clotting Factor X. In addition to reduced fertility in females, BAH null mice display several developmental defects including syndactyly, facial dysmorphology, and a mild defect in hard palate formation. The developmental defects present in BAH null mice are similar to defects observed in knock-outs and hypomorphs of the Notch ligand Serrate-2. In this work, ␤-hydroxylation of Asp residues in EGF domains is demonstrated for a soluble form of a Notch ligand, human Jagged-1. These results along with recent reports that another post-translational modification of EGF domains in Notch gene family members (glycosylation by Fringe) alters Notch pathway signaling, lends credence to the suggestion that aspartyl ␤-hydroxylation may represent another post-translational modification of EGF domains that can modulate Notch pathway signaling. Previous work has demonstrated increased levels of BAH in certain tumor tissues and a role for BAH in tumorigenesis has been proposed. The role of hydroxylase in tumor formation was tested directly by crossing BAH KO mice with an intestinal tumor model, APCmin mice. Surprisingly, BAH null/APCmin mice show a statistically significant increase in both intestinal polyp size and number when compared with BAH wild-type/APCmin controls. These results suggest that, in contrast to expectations, loss of BAH catalytic activity may promote tumor formation.

In mice, homozygosity for the Mhc haplotype H-2k is associated with increased susceptibility to spontaneous and virus-induced leukemia, lymphoma and other neoplasms in the predisposed host. The influence of the Mhc on malignant development in these models is to shorten the latency after virus inoculation. Here, we present evidence that a similar phenomenon results in early-onset of human leukemia. A molecular analysis of the MHC in 112 CML patients showed that those who developed the disease when aged less than 35 years (early-onset group) had higher homozygosity rates for the DOA1, HSP70 and C4 alleles of the DR53 group of ancestral haplotypes, for a subtype of HLA-A3, and a higher allele frequency of BfFb compared to the late-onset group. The oldest patient (n = 13) homozygous for DR53 was 52-years-old (p = 0.004), and all HLA-A3 homozygous patients (n = 4) were in the early-onset group (p = 0.01). The relative risk for early-onset CML yielded by HLA-A3 homozygosity was 17.6. The well-known serological HLA-Cw4 association was not confirmed at the DNA level and thought to be due to linkage disequilibrium with BfFb. The factor B association was sex-limited. The DR52 group haplotypes appeared to be protective. The HLA-identical sibling frequency was increased only in the early-onset group (p < 0.01). Our findings agree with the concept of an MHC influence on the development of malignancies. The similarity in the location of the susceptibility loci and the serological cross-reaction between H-2Ek and DR53 raise the possibility that the mouse and human MHC share the same leukemia susceptibility genes.

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In agreement with previous authors we found patients with Graves' disease to have an increased incidence of the DR 3 antigen. We could find no association, however, between the presence of the antigen and relapse after carbimazole treatment. A concordant HLA status and thyrotrophin receptor antibody (TRAb) index, obtained at either 6 or 12 months after the start of treatment could only predict cases of relapse and remission in a minority of patients making this of very limited clinical use. The TRAb index obtained at 12 months after the start of medical therapy could accurately predict cases of relapse and remission for the next 3 years in 24/30 patients studied.

Soybean [Glycine max (L.) Merr.] oil is one of the most consumed and highest produced vegetable oils. However, the high percentage of polyunsaturated fatty acids in soybean oil limits its stability and shelf life. Here we report the generation and characterization of a high oleic acid soybean event (305423 soybean) that has an elevated content of monounsaturated oleic acid and reduced presence of polyunsaturated linoleic acid and linolenic acid. This transgenic event was generated by the insertion of a soybean ω‐6 desaturase (FAD2) gene fragment (gm‐fad2‐1), resulting in the suppression of endogenous FAD2‐1 expression, and a modified version of the soybean acetolactate synthase (ALS) gene (gm‐hra), used as a selectable marker. Molecular characterization revealed that 305423 soybean harbors four DNA inserts containing multiple copies of complete and partial gm‐fad2‐1 gene cassettes and a single copy of the intact gm‐hra gene cassette. However, these DNA inserts seemed to be inherited together without segregation. The suppression of endogenous FAD2‐1 led to an increase of oleic acid (18:1) from 211 to 765 g kg–1 along with concurrent reduction of both linoleic acid (18:2) from 525 to 36.2 g kg–1 and linolenic acid (18:3) from 93.5 to 53.9 g kg–1 out of total fatty acyl groups. The 305423 soybean was also subjected to nutrient composition analyses and phenotypic and agronomic performance evaluations and found to be comparable to the control soybean except for the expected traits.