KRAS2 gene mutations are found in 75-90% of infiltrating pancreatic ductal adenocarcinomas but can also be present with other nonneoplastic pancreatic diseases. We recently developed a novel sensitive assay for point mutation detection, called "LigAmp", which can detect one mutant molecule in the presence of 10,000 wild-type molecules and can quantify mutant DNA over a wide dynamic range. We analyzed KRAS2 mutations in surgically-collected pancreatic duct juice samples from patients with pancreatic adenocarcinoma (n = 27) and chronic pancreatitis (n = 9). DNA sequencing demonstrated that 17 of the 27 pancreatic cancers harbored KRAS2 mutations at codon 12, including G12D (GGT → GAT), G12V (GTT), and G12R (CGT). We determined the relative amounts of each KRAS2 mutant by simultaneously quantifying wild-type and mutant KRAS2 DNA. For all pancreatic adenocarcinoma patients, the dominant KRAS2 mutation detected in the pancreatic juice corresponded to that found in the primary cancer. Mutation levels were substantially higher in patients with pancreatic cancer (0.05 to 82% of total KRAS2 molecules) compared to those with chronic pancreatitis (0 to 0.7%). Among patients with mutant KRAS2 positive cancers, all but one (94%) had mutant KRAS2 DNA concentrations of more than 0.5% in their pancreatic juice samples, whereas only 1 of 9 (11%) pancreatic juice samples from patients with chronic pancreatitis had more than 0.5% mutant KRAS2 DNA, corresponding to a sensitivity of 94% and a specificity of 89%. LigAmp quantification of mutant KRAS2 in pancreatic juice differentiates pancreatic adenocarcinoma from chronic pancreatitis, and may be a useful early detection tool for pancreatic cancer.

The essential elements of the sea urchin L. variegatus U1 snRNA promoter were mapped by microinjection of a U1 maxigene into sea urchin zygotes. Two elements are required for expression: a distal sequence element (DSE) located between -318 and -300 and a proximal sequence element (PSE) centered at -55. Removal or alteration of other sequences conserved in different sea urchin snRNA U1 genes, including deletion of all sequence between -90 and -273, did not affect the expression. Sequences around the start site were not required for expression. Deletion of nucleotides between the PSE and the start site resulted in initiation inside the U1 coding region, suggesting that the PSE determines the start site of transcription. There is no obvious similarity between the sequences required for the sea urchin U1 snRNA expression and the sequences required for the expression of other sea urchin snRNAs.

We describe the microcell fusion transfer of 100-200 kb self-replicating circular human minichromosomes from human into mouse cells. This experimental approach is illustrated through the shutting of the latent 170 kb double-stranded DNA genome from the human herpesvirus, Epstein-Barr virus, into nonpermissive rodent cells. Using this interspecies transfer strategy, circular episomes carrying 95-105 kb of human DNA were successfully established at low copy number in mouse A9 cells. Selected episomes were stably maintained for 6 months, and unselected episomes were characterized by a 95% episomal retention per cell division. The establishment of a mouse artificial episomal chromosome system should facilitate evolutionary and therapeutic studies of large human DNA in rodent genetic backgrounds.

The latent replication of oriP-based plasmids in human IR3-deleted, form, as well as a new EBNA1 isoform cloned cells depends on the viral oriP-binding transactivator from Raji. The results of a 6-month study indicate that the EBNA1. In this report, the effect of the internal repeat 3 isoforms of EBNA1 differ with respect to their efficiency of (IR3 or GlyAla repeat) domain of EBNA1 on long-term plasmid maintenance. While the EBNA-1 Raji encoding maintenance and transgene expression of OriP-based plasmid was the most stable, the oriP-based vector plasmids was examined in dividing human cells. To assess expressing the truncated EBNA1 (IR3del) gene was lost at the potential contribution of different isoforms of EBNA1 a much higher rate than those transducing full size specifically, the long-term stability of oriP-based plasmids EBNA1s. In parallel, long-term reporter gene expression in was determined after stable transfection of various CMVvarious human B cell lines was shown to persist at the driven EBNA1 genes in EBV-negative human B cells. Epihighest level with the oriP-based Raji EBNA-1 construct. some copy number was quantified using a novel sensitive These results show that the GlyAla domain can positively assay based on human mitochondrial DNA as an internal influence long-term plasmid stability and episomal transextrachromosomal control. Using this assay, the standard gene expression. B95.8-derived EBNA1 was compared with its truncated,

The formation of the 3' end of vertebrate small nuclear RNAs (snRNAs) requires that transcription initiate from an snRNA promoter. There is a loosely conserved required box 5 to 20 nucleotides (nt) (10,20,21,33). The U7 small nuclear ribonucleoprotein recognizes the downstream conserved purine-rich sequence by base pairing (3, 30).The mechanism of 3'-end formation of snRNAs in invertebrate systems has not been studied. The 3' ends of yeast snRNAs are formed properly on transcripts initiated from promoters of genes encoding mRNAs (11). In the sea urchin, there is an AG-rich sequence 3' of the snRNA genes encoding Ul, U2, and U7 snRNAs (4,8,27). This sequence is similar to the sequence 3' of the histone genes, which interacts with U7 snRNA. The structure of the sea urchin Ul snRNA promoter is similar to those of the vertebrate snRNA promoters. There are two essential sequences, the proximal sequence element, located at about -55, and a distal sequence element located at about -300 (34). The sea urchin snRNA genes are not transcribed in Xenopus oocytes (30), indicating that the promoter elements interact with different factors, although the 3' end of sea urchin U7 snRNA was formed properly when the U7 snRNA was transcribed by using a Xenopus U2 snRNA promoter (30).Here, we examine the sequence requirements for formation of the 3' end of Ul RNA in sea urchin embryos. We find that there is a bipartite signal, either part of which suffices to direct 3'-end formation. We also find that, unlike in vertebrate systems, there is efficient formation of Ul 3' ends on transcripts which are initiated by using a sea urchin histone gene promoter.

Lambda phage clones containing multiple copies of the 1.1 kb tandemly repeated unit of the sea urchin (S. purpuratus) U1 RNA genes were isolated from a gene library. The 1.1 kb repeat unit encodes a single copy of the predominant U1 RNA expressed in oocytes and embryos prior to the blastula stage. The tandem repeat unit is about 80 kb in size and is probably present one time per haploid genome as judged by pulsed-field electrophoresis of sperm DNA digested with restriction enzymes which do not cut in the repeat unit. Two of the phage contained DNA flanking the repeat unit as well as several repeat units. The tandem repeat unit ends just 3' to the U1 coding region. There is only limited homology in the 5' flanking region with U1 snRNA genes from the sea urchin L. variegatus.

The formation of the 3' end of vertebrate small nuclear RNAs (snRNAs) requires that transcription initiate from an snRNA promoter. There is a loosely conserved required box 5 to 20 nucleotides (nt) 3' of the gene. The sea urchin snRNA genes contain promoter elements different from those of the vertebrate snRNAs. They also contain a characteristic 3' 15-nt sequence which is conserved among different sea urchin snRNA genes. We used microinjection of sea urchin U1 snRNA genes into sea urchin zygotes to define the sequence requirements for U1 snRNA 3'-end formation. Surprisingly, the conserved 3' box is not required for efficient 3'-end formation in vivo. Deletion analysis reveals that the 6 nt immediately 3' of the U1 snRNA are involved in 3'-end formation. Substitution analysis revealed that either these 6 nt 3' of the U1 RNA or the conserved 3' box could direct 3'-end formation. Transcripts initiated from a histone H4 promoter formed U1 3' ends about 50% as efficiently as transcripts initiated from the U1 promoter, even when the U1 end was placed in tandem with a histone 3'-processing signal, suggesting that transcription from an snRNA promoter is not necessary for formation of the 3' end of sea urchin U1 snRNA.

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