The poly(A) tract Since post-transcriptional events may represent an important control mechanism in the regulation of genetic expression, we have investigated the possibility of methylation as an additional post-transcriptional modification of mRNA. Earlier work with bacterial and phage mRNA produced strong evidence for the essential absence of methylation in these systems, being no higher than one per 3500 nucleotides (8). Other early studies with mammalian heterogeneous nuclear RNA (HnRNA) indicated that methylation was either nonexistent or very low (9, 10). The discovery of poly-(A) has now made it possible to obtain pure mRNA fractions through affinity chromatography and, therefore, to search for low levels of methylation in mRNA without interference from rRNA contamination. Recently Perry and Kelley reported the existence of methylation in mouse L cell mRNA at about one-sixth the level found in rRNA, and both base and ribose methylations were found (11). This paper reports the existence of methylated nucleosides in the mRNA of Novikoff hepatoma cells and identifies the unique distribution of methylated moieties. A preliminary report of these results has appeared elsewhere (12). METHODSCell Culture and Labeling. The NlS1 strain of Novikoff hepatoma cells was grown in culture in Swimm's S-77 medium supplemented with 4 mM glutamine and 10% (v/v) dialyzed calf serum (13

The high frequency of carriers of mutations in GJB2 (4.76 percent) predicts a prevalence of 1 deaf person among 1765 people, which may account for the majority of cases of nonsyndromic recessive deafness in the Ashkenazi Jewish population. Conservation of the haplotype flanking the 167delT mutation suggests that this allele has a single origin, whereas the multiple haplotypes with the 30delG mutation suggest that this site is a hot spot for recurrent mutations.

An analysis of the methylated constituents of L cell mRNA by a combination of chromatographic methods and enzymatic treatments indicates that they comprise both 2'-O-methyl nucleosides and N6-methyl adenine, and/or 1-methyl adenine, and suggests that the 2'-O-methyl nucleotides, Ym, are part of an unusual class of sequences forming the 5' terminus of mRNA. These sequences seem to contain two 2'-O-methyl residues and a terminal residue that is not phosphorylated but, nevertheless, is blocked with respect to polynucleotid kinase reactivity. A strong candidate is a sequence of the type XppY1mpY2mpZp..., where X represents a blocking group which is itself occasionally methylated. The sequences isolated from total poly(A)+ mRNA contain all four species of 2'-O-methylated nucleoside, indicating some variability among different mRNA species. The methylated sequences do not appear to be enriched in the mRNA which hybridizes with repetitive DNA. The average L cell mRNA molecule also contains three residues of N6-methyl adenine. These residues are not part of the poly(A) segment, but appear to be located internal to the poly(A) near the 3' end of the mRNA molecules.

␤cyto-Actin and ␥cyto-actin are ubiquitous proteins thought to be essential building blocks of the cytoskeleton in all non-muscle cells. Despite this widely held supposition, we show that ␥cyto-actin null mice (Actg1 ؊/؊ ) are viable. However, they suffer increased mortality and show progressive hearing loss during adulthood despite compensatory up-regulation of ␤cyto-actin. The surprising viability and normal hearing of young Actg1 ؊/؊ mice means that ␤cyto-actin can likely build all essential non-muscle actin-based cytoskeletal structures including mechanosensory stereocilia of hair cells that are necessary for hearing. Although ␥cyto-actin-deficient stereocilia form normally, we found that they cannot maintain the integrity of the stereocilia actin core. In the wild-type, ␥cyto-actin localizes along the length of stereocilia but re-distributes to sites of F-actin core disruptions resulting from animal exposure to damaging noise. In Actg1 ؊/؊ stereocilia similar disruptions are observed even without noise exposure. We conclude that ␥cyto-actin is required for reinforcement and long-term stability of F-actin-based structures but is not an essential building block of the developing cytoskeleton.actin ͉ cytoskeleton ͉ hearing T here are six genes encoding six vertebrate actins that are classified according to where they are predominately expressed. ␣ skeletal -Actin, ␣ smooth -actin, ␣ cardiac -actin, and ␥ smoothactin are primarily found in muscle cells, whereas cytoplasmic ␤ cyto -actin and ␥ cyto -actin are ubiquitously and highly expressed in non-muscle cells, as reviewed elsewhere (1). Athough ␤ cytoactin and ␥ cyto -actin differ at only four biochemically similar amino acid residues in their N-termini, several lines of evidence suggest that each protein is functionally distinct. The amino acid sequences of ␤ cyto -and ␥ cyto -actin are each exactly conserved among avian and mammalian species. In addition, ␤ cyto -and ␥ cyto -actin proteins are differentially localized (2-5) and posttranslationally modified (6). Finally, although dominant missense mutations in ACTB encoding ␤ cyto -actin are associated with syndromic phenotypes including severe developmental malformations and bilateral deafness (7), humans carrying a variety of dominant missense mutations in ACTG1 develop postlingual nonsyndromic progressive hearing loss (DFNA20, OMIM 604717) (8-11).␥ cyto -Actin is widely expressed in the inner ear sensory epithelial cells on which mammalian hearing depends. These cells are organized in rows along the cochlea length: one row of inner hair cells (IHCs) and three rows of outer hair cells (OHCs) (Fig. 2A). IHCs function as auditory receptors, converting sound energy into neuronal signals, whereas OHCs enhance sensitivity to sound stimuli, as reviewed elsewhere (12). The apical surface of a hair cell is topped with actin-rich microvilli-derived protrusions termed stereocilia, which deflect in response to sound stimuli, initiating mechanoelectrical transduction (Fig. 2B). ␤ cyto -and ␥ cyto -Actin are both thou...

KOH digestion of methyl-labeled poly(A)+ mRNA purified by (dT)-cellulose chromatography produced mononucleotide and multiple peaks of a large oligonucleotide (-6 to -8 charge) when separated on the basis of charge by Pellionex-WAX high-speed liquid chromatography in 7 M urea. Heat denaturation of the RNA before application to (dT)-cellulose was required to release contaminants (mostly 18S rRNA) that persisted even after repeated binding to (dT)-cellulose at room temperature. Analysis of the purified poly(A)+ mRNA by enzyme digestion, acid hydrolysis, and a variety of chromatographic techniques has shown that the monucleotide (53%) is due entirely to N6-methyladenosine. The large oligonucleotides (47%) were found to contain 7-methylguanosine and the 2'-0-methyl derivatives of all four nucleosides. No radioactivity was found associated with the poly(A) segment. Periodate oxidation of the mRNA followed by beta elimination released only labeled 7-methylguanine consistent with a blocked 5' terminus containing an unusual 5'-5' bond. Alkaline phosphatase treatment of intact mRNA had no effect on the migration of the KOH produced oligonucleotides on Pellionex-WAX. When RNA from which 7-methylguanine was removed by beta elimination was used for the phosphatase treatment, distinct dinucleotides (NmpNp) and trinucleotides (NmpNmpNp) occurred after KOH hydrolysis and Pellionex-WAX chromatography. Thus Novikoff hepatoma poly(A)+ mRNA molecules can contain either one or two 2'-0-methylnucleotides linked by a 5'-5' bond to a terminal 7-methylguanosine and the 2'-0-methylation can occur with any of the four nucleotides. The 5' terminus may be represented by m7G5'ppp5' (Nmp)lor2Np, a general structure proposed earlier as a possible 5' terminus for all eucaryotic mRNA molecules (Rottman, F., Shatkin, A., and Perry, R. (1974), Cell 3, 197). The composition analyses indicate that there are 3.0 N6-methyladenosine residues, 1.0 7-methylguanosine residue, and 1.7 2'-0-methylnucleoside residues per average mRNA molecule.

Age-related hearing loss (presbycusis) is a significant problem in the population. The genetic contribution to age-related hearing loss is estimated to be 40%-50%. Gene mutations that cause nonsyndromic progressive hearing loss with early onset may provide insight into the etiology of presbycusis. We have identified four families segregating an autosomal dominant, progressive, sensorineural hearing loss phenotype that has been linked to chromosome 17q25.3. The critical interval containing the causative gene was narrowed to approximately 2 million bp between markers D17S914 and D17S668. Cochlear-expressed genes were sequenced in affected family members. Sequence analysis of the gamma-actin gene (ACTG1) revealed missense mutations in highly conserved actin domains in all four families. These mutations change amino acids that are conserved in all actins, from protozoa to mammals, and were not found in >100 chromosomes from normal hearing individuals. Much of the specialized ultrastructural organization of the cells in the cochlea is based on the actin cytoskeleton. Many of the mutations known to cause either syndromic or nonsyndromic deafness occur in genes that interact with actin (e.g., the myosins, espin, and harmonin). The mutations we have identified are in various binding domains of actin and are predicted to mildly interfere with bundling, gelation, polymerization, or myosin movement and may cause hearing loss by hindering the repair or stability of cochlear cell structures damaged by noise or aging. This is the first description of a mutation in cytoskeletal, or nonmuscle, actin.

␤-and ␥-nonmuscle actins differ by 4 amino acids at or near the N terminus and distant from polymerization interfaces. ␤-Actin contains an Asp 1 -Asp 2 -Asp 3 and Val 10 whereas ␥-actin has a Glu 1 -Glu 2 -Glu 3 and Ile 10 . Despite these small changes, conserved across mammals, fish, and birds, their differential localization in the same cell suggests they may play different roles reflecting differences in their biochemical properties. To test this hypothesis, we established a baculovirus-driven expression system for producing these actins in isoform-pure populations although contaminated with 20 -25% insect actin. Surprisingly, Ca-␥-actin exhibits a slower monomeric nucleotide exchange rate, a much longer nucleation phase, and a somewhat slower elongation rate than ␤-actin. In the Mg-form, this difference between the two is much smaller. Ca-␥-actin depolymerizes half as fast as does ␤-actin. Mixing experiments with Caactins reveal the two will readily co-polymerize. In the Ca-form, phosphate release from polymerizing ␤-actin occurs much more rapidly and extensively than polymerization, whereas phosphate release lags behind polymerization with ␥-actin. Phosphate release during treadmilling is twice as fast with ␤-as with ␥-actin. With Mg-actin in the initial stages, phosphate release for both actins correlates much more closely with polymerization. Calcium bound in the high affinity binding site of ␥-actin may cause a selective energy barrier relative to ␤-actin that retards the equilibration between G-and F-monomer conformations resulting in a slower polymerizing actin with greater filament stability. This difference may be particularly important in sites such as the ␥-actin-rich cochlear hair cell stereocilium where local mM calcium concentrations may exist.

BACKGROUND-Recent studies have suggested that child ADHD and its symptom domains are related to blood lead level, even at background exposure levels typical in western countries. However, recent studies disagreed as to whether lead was related to inattention or hyperactivityimpulsivity within the ADHD domain. More definitive evaluation of these questions was sought.