Requirement of the nicotinic acetylcholine receptor β2 subunit for the anatomical and functional development of the visual system

In the mammalian visual system the formation of eye-specific layers at the thalamic level depends on retinal waves of spontaneous activity, which rely on nicotinic acetylcholine receptor activation. We found that in mutant mice lacking the β2 subunit of the neuronal nicotinic receptor, but not in mice lacking the α4 subunit, retinofugal projections do not segregate into eye-specific areas, both in the dorso-lateral geniculate nucleus and in the superior colliculus. Moreover, β2−/− mice show an expansion of the binocular subfield of the primary visual cortex and a decrease in visual acuity at the cortical level but not in the retina. We conclude that the β2 subunit of the nicotinic acetylcholine receptor is necessary for the anatomical and functional development of the visual system.

Development of an in vitro mRNA decay system for Escherichia coli: Poly(A) polymerase I is necessary to trigger degradation

Using a novel Escherichia coli in vitro decay system in which polysomes are the source of both enzymes and mRNA, we demonstrate a requirement for poly(A) polymerase I (PAP I) in mRNA turnover. The in vitro decay of two different mRNAs (trxA and lpp) is triggered by the addition of ATP only when polysomes are prepared from a strain carrying the wild-type gene for PAP I (pcnB+). The relative decay rates of these two messages are similar in vitro and in vivo. Poly(A) tails are formed on both mRNAs, but no poly(A) tails are detected on the 3′ end of mature 23S rRNA. The size distribution of poly(A) tails generated in vitro, averaging 50 nt in length, is comparable to that previously reported in vivo. PAP I activity is associated exclusively with the polysomes. Exogenously added PAP I does not restore mRNA decay to PAP I− polysomes, suggesting that, in vivo, PAP I may be part of a multiprotein complex. The potential of this in vitro system for analyzing mRNA decay in E. coli is discussed.

A small nucleolar RNA requirement for site-specific ribose methylation of rRNA in Xenopus

Vertebrate cells contain a large number of small nucleolar RNA (snoRNA) species, the vast majority of which bind fibrillarin. Most of the fibrillarin-associated snoRNAs can form 10- to 21-nt duplexes with rRNA and are thought to guide 2′-O-methylation of selected nucleotides in rRNA. These include mammalian UHG (U22 host gene)-encoded U25–U31 snoRNAs. We have characterized two novel human snoRNA species, U62 and U63, which similarly exhibit 15- (with one interruption) and 12-nt complementarities and are therefore predicted to direct 2′-O-methylation of A590 in 18S and A4531 in 28S rRNA, respectively. To establish the function of antisense snoRNAs in vertebrates, we exploited the Xenopus oocyte system. Cloning of the Xenopus U25–U31 snoRNA genes indicated that they are encoded within multiple homologs of mammalian UHG. Depletion of U25 from the Xenopus oocyte abolished 2′-O-methylation of G1448 in 18S rRNA; methylation could be restored by injecting either the Xenopus or human U25 transcript into U25-depleted oocytes. Comparison of Xenopus and human U25 sequences revealed that only boxes C, D, and D′, as well as the 18S rRNA complement, were invariant, suggesting that they may be the only elements required for U25 snoRNA stability and function.

Requirement of the Lec35 Gene for All Known Classes of Monosaccharide-P-Dolichol-dependent Glycosyltransferase Reactions in Mammals

The Lec35 gene product (Lec35p) is required for utilization of the mannose donor mannose-P-dolichol (MPD) in synthesis of both lipid-linked oligosaccharides (LLOs) and glycosylphosphatidylinositols, which are important for functions such as protein folding and membrane anchoring, respectively. The hamster Lec35 gene is shown to encode the previously identified cDNA SL15, which corrects the Lec35 mutant phenotype and predicts a novel endoplasmic reticulum membrane protein. The mutant hamster alleles Lec35.1 and Lec35.2 are characterized, and the human Lec35 gene (mannose-P-dolichol utilization defect 1) was mapped to 17p12-13. To determine whether Lec35p was required only for MPD-dependent mannosylation of LLO and glycosylphosphatidylinositol intermediates, two additional lipid-mediated reactions were investigated: MPD-dependent C-mannosylation of tryptophanyl residues, and glucose-P-dolichol (GPD)-dependent glucosylation of LLO. Both were found to require Lec35p. In addition, the SL15-encoded protein was selective for MPD compared with GPD, suggesting that an additional GPD-selective Lec35 gene product remains to be identified. The predicted amino acid sequence of Lec35p does not suggest an obvious function or mechanism. By testing the water-soluble MPD analog mannose-β-1-P-citronellol in an in vitro system in which the MPD utilization defect was preserved by permeabilization with streptolysin-O, it was determined that Lec35p is not directly required for the enzymatic transfer of mannose from the donor to the acceptor substrate. These results show that Lec35p has an essential role for all known classes of monosaccharide-P-dolichol-dependent reactions in mammals. The in vitro data suggest that Lec35p controls an aspect of MPD orientation in the endoplasmic reticulum membrane that is crucial for its activity as a donor substrate.

In vivo requirement for RecJ, ExoVII, ExoI, and ExoX in methyl-directed mismatch repair

Biochemical studies with model DNA heteroduplexes have implicated RecJ exonuclease, exonuclease VII, exonuclease I, and exonuclease X in Escherichia coli methyl-directed mismatch correction. However, strains deficient in the four exonucleases display only a modest increase in mutation rate, raising questions concerning involvement of these activities in mismatch repair in vivo. The quadruple mutant deficient in the four exonucleases, as well as the triple mutant deficient in RecJ exonuclease, exonuclease VII, and exonuclease I, grow poorly in the presence of the base analogue 2-aminopurine, and exposure to the base analogue results in filament formation, indicative of induction of SOS DNA damage response. The growth defect and filamentation phenotypes associated with 2-aminopurine exposure are effectively suppressed by null mutations in mutH, mutL, mutS, or uvrD/mutU, which encode activities that act upstream of the four exonucleases in the mechanism for the methyl-directed reaction that has been proposed based on in vitro studies. The quadruple exonuclease mutant is also cold-sensitive, having a severe growth defect at 30°C. This phenotype is suppressed by a uvrD/mutU defect, and partially suppressed by mutH, mutL, or mutS mutations. These observations confirm involvement of the four exonucleases in methyl-directed mismatch repair in vivo and suggest that the low mutability of exonuclease-deficient strains is a consequence of under recovery of mutants due to a reduction in viability and/or chromosome loss associated with activation of the mismatch repair system in the absence of RecJ exonuclease, exonuclease VII, exonuclease I, and exonuclease X.

Human general transcription factor TFIIA: characterization of a cDNA encoding the small subunit and requirement for basal and activated transcription.

The human general transcription factor TFIIA is one of several factors involved in specific transcription by RNA polymerase II, possibly by regulating the activity of the TATA-binding subunit (TBP) of TFIID. TFIIA purified from HeLa extracts consists of 35-, 19-, and 12-kDa subunits. Here we describe the isolation of a cDNA clone (hTFIIA gamma) encoding the 12-kDa subunit. Using expression constructs derived from hTFIIA gamma and TFIIA alpha/beta (which encodes a 55-kDa precursor to the alpha and beta subunits of natural TFIIA), we have constructed a synthetic TFIIA with a polypeptide composition similar to that of natural TFIIA. The recombinant complex supports the formation of a DNA-TBP-TFIIA complex and mediates both basal and Gal4-VP16-activated transcription by RNA polymerase II in TFIIA-depleted nuclear extracts. In contrast, TFIIA has no effect on tRNA and 5S RNA transcription by RNA polymerase III in this system. We also present evidence that both the p55 and p12 recombinant subunits interact with TBP and that the basic region of TBP is critical for the TFIIA-dependent function of TBP in nuclear extracts.