A U1 small nuclear ribonucleoprotein particle with altered specificity induces alternative splicing of an adenovirus E1A mRNA precursor.

We have altered the specificity of U1 small nuclear RNA by replacing its 5' splice site recognition sequence (nucleotides 3 to 11) with sequences complementary to other regions of either the adenovirus E1A or the rabbit beta-globin mRNA precursor. We then used a HeLa cell transient expression assay to test whether such altered U1 small nuclear ribonucleoprotein particles (snRNPs) could interfere with splicing of the targeted mRNA precursors. The altered U1 snRNPs were able to cause novel splicing of the E1A mRNA precursor, minor changes in the ratio of E1A 12 to 13S mRNAs, and modest nuclear accumulation of beta-globin mRNA precursors with either one of the two introns removed. Most of the altered U1 snRNPs did not affect the level of mature cytoplasmic mRNA significantly, but in one case an altered U1 snRNP (alpha 1) whose intended target was located downstream from the adenovirus E1A 12S 5' splice site was able to reduce the level of cytoplasmic 12S mRNA by approximately 60% and that of 13S mRNA by 90%. This alpha 1 snRNP induced an additional E1A splice, resulting in the appearance of 10 and 11S E1A mRNAs normally found only late in adenovirus infection. Thus, a trans-acting factor can induce alternative splicing. Surprisingly, the effects of alpha 1 on E1A splicing were not abolished by deleting the intended target sequence on the mRNA precursor.

Sequence of a human E2F-4 pseudogene with shortened AGC trinucleotide repeats.

We have isolated a human genomic DNA fragment of 0.7 kb by polymerase chain reaction (PCR) using human E2F-4 gene primers. Sequence analysis indicates that this DNA comes from a processed E2F-4 pseudogene. Its sequence is 75% identical to the human E2F-4 cDNA, and bears several deletions and insertions. A 30 bp deletion shortens the 13 AGC repeats in the E2F-4 gene by 10 repeats. The sequence data also suggest that another 65 bp deletion and some small insertions may be generated by replication errors on template pairing.

Genetic analysis of the role of human U1 snRNA in mRNA splicing: I. Effect of mutations in the highly conserved stem-loop I of U1.

The 5' splice site mutation known as hr440 can be suppressed efficiently in vivo by a compensatory base change in U1 small nuclear RNA (snRNA). We have now begun a second-site reversion analysis of this suppressor U1-4u snRNA (which has a C----U change at position 4) to identify U1 nucleotides that are essential for mRNA splicing. Point mutations in U1-4u that disrupt the structure of stem-loop I or alter phylogenetically conserved nucleotides within the loop cause loss of suppression. The level of suppressor activity observed for most mutants correlated with the abundance of the corresponding suppressor RNA, suggesting that mutations in stem-loop I cause loss of suppression by destabilizing U1 snRNA or the U1 snRNP (small nuclear ribonucleoprotein particle). We favor the interpretation that incompletely or improperly assembled U1 snRNPs are unstable, because two severe point mutations in stem-loop I were found to decrease the binding of U1 snRNP-specific proteins in vitro. In a separate set of experiments, we found that increasing the distance between stem-loop I and the 5' end of U1 snRNA also inhibited suppression but did not affect assembly or stability of the U1 snRNP. This suggests that the relationship between the 5' splice site and the body of the U1 snRNP is important for mRNA splicing.

Sequences required for 3' end formation of human U2 small nuclear RNA.

Xenopus oocytes injected with human U2 snRNA genes synthesize mature U2 as well as a U2 precursor with about 10 extra 3' nucleotides (human pre-U2 RNA). Formation of the pre-U2 3' end requires a downstream element located between position +16 and +37 in the U2 3'-flanking sequence. The distance between this element and the U2 coding region can be increased without affecting formation of the pre-U2 3' end. When the natural sequence surrounding the pre-U2 3' end is changed, novel 3' ends are still generated within a narrow range upstream from the element. The 3' terminal stem-loop of U2 snRNA is not required for pre-U2 3' end formation. A sequence within the 3' element (GTTTN0-3AAAPuNNAGA) is conserved among snRNA genes transcribed by RNA polymerase II. Our results suggest that the 3' ends of pre-U2 RNA and histone mRNA may be generated by related but distinct RNA processing mechanisms.

Stable expression of functional human cytomegalovirus immediate-early proteins IE1 and IE2 in HeLa cells.

The major immediate-early (IE) genes 1 and 2 of human cytomegalovirus (HCMV) encode proteins that regulate the expression of HCMV genes as well as some other viral and cellular genes. In order to study the expression and function of these IE gene products, we established several HeLa cell lines that stably expressed the 68-kD IE1 protein, the 82-kD IE2 protein, or both proteins. The IE proteins expressed in these cell lines were biologically active, as shown by transient chloramphenicol acetyltransferase assays. Transcription from the major IE promoter was augmented in the IE1-expressing cells, while transcription from the HCMV early gene UL84 promoter was activated in the IE2-expressing cells. In addition, we found that the IE2-expressing cells established colonies in soft agarose more efficiently than the parental HeLa and the IE1-expressing cells. Furthermore, expression of both the IE1 and IE2 proteins was increased by treatment of these cell lines with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. Thus, our cell lines provide a useful system to study the regulation of IE gene expression in human cells as well as to study transaction by HCMV IE proteins on various viral and cellular genes.

Recovery of Salmonella by using selenite brilliant green sulfa enrichment broth.

The efficacy and sensitivity of selenite brilliant green sulfa enrichment (SBG) broth for the isolation of Salmonella from fecal specimens were evaluated by using both clinical and artificially infected (artificial) fecal specimens. An examination of 1,588 clinical fecal specimens found Salmonella in 296 specimens, including 89 cases detected by the direct-plating xylose-lysine-desoxycholate method and an additional 207 cases detected after enrichment with SBG broth. Therefore, the recovery of Salmonella with SBG broth is increased 3.3-fold over that by the direct-plating method alone. Furthermore, the isolation rate of Salmonella is higher when using SBG broth than when using gram-negative (GN) broth or GN broth supplemented with sodium selenite. To determine the sensitivity for the recovery of Salmonella, artificial specimens containing various amounts of Salmonella were prepared and analyzed. The results indicated that the sensitivity is also higher with SBG broth than with GN broth. Moreover, the optimal incubation period for SBG broth can be extended to 24 h. In conclusion, the SBG enrichment method provides a higher recovery rate of Salmonella from fecal specimens.