Hypomorphic mutation in hnRNP U results in post-implantation lethality.

The present study characterized an embryonic lethal mutation induced by insertion of the U3Neo gene trap retrovirus into an intron of the gene encoding heterogeneous ribonuclear protein U (Hnrnpu), which maps to the distal arm of mouse chromosome 1. Murine hnRNP U was found to be identical to the human protein at all but one of 341 amino acid residues. Embryos homozygous for the provirus showed obvious abnormalities after 6.5 days of development (E6.5) and were resorbed by E10.5. Expression of the inserted neomycin-resistance gene involved alternative splicing to a cryptic 3' splice site located in the neomycin resistance gene resulting in a hypomorphic mutation. Homozygous mutant cell lines isolated from preimplantation blastocysts expressed hnRNP U transcripts at levels 2 to 5 times lower than wild-type cells, suggesting that nearly wild-type levels of hnRNP U are required for embryonic development.

Estrogen receptor-alpha messenger RNA variants that lack exon 5 or exon 7 are coexpressed with wild-type form in human endometrium during all phases of the menstrual cycle.

OBJECTIVE: We have assessed the expression levels of messenger RNA for estrogen receptor-alpha and splice variants lacking exon 5 or exon 7 that presumably exert dominant positive (splice variants lacking exon 5) and negative (splice variants lacking exon 7) effects, respectively, on estrogen responses in the human endometrium. STUDY DESIGN: This was a prospective study that was conducted at an academic community-based hospital. The patients, aged 18 to 40 years, underwent hysterectomy for benign gynecologic causes. Eighty-one endometrial specimens (46 proliferative, 35 secretory) were analyzed with the use of reverse transcription-polymerase chain reaction assay for the messenger RNA levels of estrogen receptor-alpha, and splice variants lacking exon 5 and exon 7. RESULTS: Wild-type estrogen receptor-alpha and splice variants splice variants lacking exon 5 and lacking exon 7 messenger RNAs were detected in all endometrial specimens throughout the menstrual cycle. In addition, a double-splice estrogen receptor-alpha messenger RNA variant (splice variants lacking exon 5 and exon 7) was detected at constant low levels of expression. Semiquantitative analysis showed higher levels of estrogen receptor-alpha messenger RNA in the early and mid proliferative endometrial phases than in late proliferative and secretory endometrium ( P <.05). The splice variant lacking exon 7 messenger RNA expression level was about 10-fold higher than the splice variant lacking exon 5 messenger RNA relative to wild-type estrogen receptor-alpha messenger RNA ( P <.001). The expression of splice variants lacking exon 5 compared with wild-type estrogen receptor-alpha messenger RNA is relatively constant throughout endometrial development. In contrast, an examination of the ratio of the levels of splice variants lacking exon 7 to wild-type estrogen receptor-alpha messenger RNA indicated a small, but significantly higher, splice variant lacking exon 7 level in the mid secretory phase (postovulatory days 5-8) than the mid proliferative and early secretory phases ( P <.05). CONCLUSION: We found no evidence of differential coexpression of the positive dominant estrogen receptor variant, splice variants lacking exon 5, with wild-type estrogen receptor-alpha. We did find that the dominant negative splice variant lacking exon 7 was slightly increased relative to wild-type estrogen receptor-alpha in the postovulatory phase. Future investigation is required to suggest the biologic significance of the observed increased relative expression of the splice variants lacking exon 7 in secretory endometrium and to determine the function of splice variants lacking exon 5 and splice variants lacking exon 7.

Activation of cryptic 3' splice sites within introns of cellular genes following gene entrapment.

Gene trap vectors developed for genome-wide mutagenesis can be used to study factors governing the expression of exons inserted throughout the genome. For example, entrapment vectors consisting of a partial 3'-terminal exon [i.e. a neomycin resistance gene (Neo), a poly(A) site, but no 3' splice site] were typically expressed following insertion into introns, from cellular transcripts that spliced to cryptic 3' splice sites present either within the targeting vector or in the adjacent intron. A vector (U3NeoSV1) containing the wild-type Neo sequence preferentially disrupted genes that spliced in-frame to a cryptic 3' splice site in the Neo coding sequence and expressed functional neomycin phosphotransferase fusion proteins. Removal of the cryptic Neo 3' splice site did not reduce the proportion of clones with inserts in introns; rather, the fusion transcripts utilized cryptic 3' splice sites present in the adjacent intron or generated by virus integration. However, gene entrapment with U3NeoSV2 was considerably more random than with U3NeoSV1, consistent with the widespread occurrence of potential 3' splice site sequences in the introns of cellular genes. These results clarify the mechanisms of gene entrapment by U3 gene trap vectors and illustrate features of exon definition required for 3' processing and polyadenylation of cellular transcripts.

Cardiac UCP2 expression and myocardial oxidative metabolism during acute septic shock in the rat.

UNLABELLED: Septic shock decreases cardiac hydraulic work relative to the rate of myocardial oxygen consumption, causing decreased mechanical efficiency (hydraulic work/myocardial oxygen consumption). This study tested whether the mitochondrial uncoupling protein UCP2 was responsible for decreased cardiac mechanical efficiency after polymicrobial septic shock. Sepsis was initiated in ketamine/xylazine-anesthetized rats by cecal ligation and puncture (CLP). Steady-state mRNA content was quantified by Northern blot analysis, and protein content was estimated by western blot. Additional hearts were removed after 12 h and perfused in working mode to measure work (mmHg x mL/min/100 g dry wt) and efficiency (CE = work/oxygen consumption, %). The 72-h mortality rate was 80%, and deaths occurred between 12-32 h. Cardiac work (152 +/- 15, shock vs. 235 +/- 16, control; P < 0.05) and cardiac efficiency (4.0 +/- 0.4 vs. 5.6 +/- 0.3; P < 0.05) were significantly decreased when hearts were isolated 12 h after CLP. Myocardial UCP2 mRNA expression was increased by 52% (12 h) compared with control hearts; however, there was no detectable UCP2 protein in mitochondria isolated from either control or septic hearts. CONCLUSIONS: Although polymicrobial sepsis decreased cardiac mechanical efficiency and increased UCP-2 expression coincident with premortal hypothermia, we did not detect any evidence of UCP-2 protein in septic heart muscle. These data argue against the hypothesis that UCP-2 causes decreased cardiac mechanical efficiency in septic shock.