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The generation of Chinese hamster ovary cell lines that express assembly defective forms of beta-tubulin were isolated using selections based on reversion of conditional lethal or drug resistance phenotypes. Two such cell lines, D2 and 6H3, were chosen for further characterization because they contain beta-tubulin polypeptides that exhibit decreases in apparent molecular weight on two-dimensional gel electrophoresis. Analysis of the nucleic acid from these cell lines using both Southern and Northern procedures suggests a deletion in one of the beta-tubulin genes in each cell line. Localization of the missing sequence in D2 was first determined by tryptic peptide mapping by high performance liquid chromatography. Subsequently, the assignment was confirmed by constructing appropriate subclones of a wild type Chinese hamster ovary beta-tubulin cDNA for Southern analysis to demonstrate a failure to recognize characteristic hybridization patterns of the mutant tubulin gene. In the other revertant, 6H3, the deletion was detected on a Northern blot by differential hybridization of a 3′ fragment of the cDNA to the beta-tubulin messages. The results indicate that D2 has an internal deletion whose approximate limits extend from amino acid residues 250 through 345. Cell line 6H3 has a deletion that begins near amino acid residue 330 and extends into the 3′-untranslated region of the gene.

Eight strains of Chinese hamster ovary (CHO) cells having an assembly-defective beta-tubulin were found among revertants of strain Cmd 4, a mutant with a conditional lethal mutation in a beta-tubulin gene (F. Cabral, M. E. Sobel, and M. M. Gottesman, Cell 20:29-36, 1980). The altered beta-tubulins in these strains have electrophoretically silent alterations or, in some cases, an increase or a decrease in apparent molecular weight based on their migration in two-dimensional gels. The identity of these variant proteins as beta-tubulin was confirmed by peptide mapping, which also revealed the loss of distinct methionine-containing peptides in the assembly-defective beta-tubulins of lower apparent molecular weight. The altered mobility of these beta-tubulin polypeptides was not the result of a posttranslational modification, since the altered species could be labeled in very short incubations with [35S]methionine and were found among in vitro-translated polypeptides by using purified mRNA. In at least one strain, an altered DNA restriction fragment could be demonstrated, suggesting that an alteration occurred in one of the structural genes for beta-tubulin. Assembly-defective beta-tubulin was unstable and turned over with a half-life of only 1 to 2 h in exponentially growing cells. This rapid degradation of a tubulin gene product resulted in approximately 30% lower steady-state levels of both alpha- and beta-tubulin yet did not affect the growth rate of the cells or the distribution of the microtubules as judged by immunofluorescence microscopy. These results argue that CHO cells possess a beta-tubulin gene product that is not essential for survival.

We have used fluorescence in situ hybridization on interphase nuclei of normal female cells to compare the replication timing patterns of genes on the human X chromosome that are known to escape X inactivation with those that are inactivated. By this procedure it was possible not only to determine the relative time of replication of the earlier-replicating allele for different loci but also to estimate the degree of asynchrony of replication of the two alleles for each individual locus. Loci such as HPRT and FRAXA, which are normally inactivated, displayed a high degree of replication asynchrony, whereas loci that are not inactivated (ZFX and RPS4X) were found to replicate very synchronously. Interestingly, examination of XIST, which is expressed only from the inactive X chromosome, by this procedure revealed that it also replicated asynchronously, with the expressed copy apparently replicating first. Therefore, by examining different loci from the X chromosome it was determined that there is a strict correlation between the expression and relative time of replication of individual genes.

We describe a simple electro-optically activated external-cavity diode laser designed to provide high-speed, high-coherence tuning over gigahertz frequency ranges. Tuning as fast as 23 GHz/µs is demonstrated. Coherence measurements indicate transform-limited quiescent laser linewidth in observation windows as wide as ?100 µs and transform-limited chirps. A self-heterodyne, cross-correlation-based coherence diagnostic is developed for characterizing phase coherence during high-speed chirps.

We propose and demonstrate a novel frequency-stabilization scheme that provides linewidth narrowing and at the same time is compatible with high-speed frequency agility. The method relies on sensing and control of a heterodyne beat signal derived from a fiber interferometer and functions in the absence of a fixed reference frequency. Our demonstration utilizes a short-external-cavity diode laser equipped with an intracavity electro-optic crystal for frequency correction. The stabilization method is shown to suppress impressed laser frequency modulation by nearly 2 orders of magnitude. Without further modification of our scheme we also demonstrate laser frequency tuning control.