Arginine deprivation in KB cells: I. Effect on cell cycle progress

When exponentially growing KB cells were deprived of arginine, cell multiplication ceased after 12 h but viability was maintained throughout the experimental period (42-48 h). Although tritiated thymidine ([(3)H]TdR) incorporation into acid-insoluble material declined to 5 percent of the initial rate, the fraction of cells engaged in DNA synthesis, determined by autoradiography, remained constant throughout the starvation period and approximately equal to the synthesizing fraction in exponentially growing controls (40 percent). Continous [(3)H]TdR-labeling indicated that 80 percent of the arginine-starved cells incorporated (3)H at some time during a 48-h deprivation period. Thus, some cells ceased DNA synthesis, whereas some initially nonsynthesizing cells initiated DNA synthesis during starvation. Flow microfluorometric profiles of distribution of cellular DNA contents at the end of the starvation period indicated that essentially no cells had a 4c or G2 complement. If arginine was restored after 30 h of starvation, cultures resumed active, largely asynchronous division after a 16-h lag. Autoradiographs of metaphase figures from cultures continuously labeled with [(3)H]TdR after restoration indicated that all cells in the culture underwent DNA synthesis before dividing. It was concluded that the majority of cells in arginine-starved cultures are arrested in neither a normal G1 nor G2. It is proposed that for an exponential culture, i.e. from most positions in the cell cycle, inhibition of cell growth after arginine with withdrawal centers on the ability of cells to complete replication of their DNA.     

A study of non-metric (qualitative) variation in Gujarati crania

Three hundred and seventy adult skulls (284 crania of unknown sex, 58 males and 28 females) from Gujarat State of India were examined for the incidence of non-metric variants and compared with other populations to establish the distance between them. In general the Gujarati incidences are of similar order to those in other series. The mean measures of divergence between Gujarati and other populations were all statistically significant (P less than 0.001). The Gujarati differed most from Australian Aborigines, but only slightly from the Burma, Punjab and Egypt samples. From the same material side and sex dimorphism was also tested to ascertain that how far sides and sexes can be pooled in Indian sample for making comparison between populations. In Gujarati population out of 22 cranial variants only four show sex difference and in case of bilateral traits, none of the variant has shown significant (P less than 0.05) side to side difference.     

Targeting of NH2-terminal–processed Microsomal Protein to Mitochondria: A Novel Pathway for the Biogenesis of Hepatic Mitochondrial P450MT2

Cytochrome P4501A1 is a hepatic, microsomal membrane–bound enzyme that is highly induced by various xenobiotic agents. Two NH₂-terminal truncated forms of this P450, termed P450MT2a and MT2b, are also found localized in mitochondria from β-naphthoflavone–induced livers. In this paper, we demonstrate that P4501A1 has a chimeric NH₂-terminal signal that facilitates the targeting of the protein to both the ER and mitochondria. The NH₂-terminal 30–amino acid stretch of P4501A1 is thought to provide signals for ER membrane insertion and also stop transfer. The present study provides evidence that a sequence motif immediately COOH-terminal (residues 33–44) to the transmembrane domain functions as a mitochondrial targeting signal under both in vivo and in vitro conditions, and that the positively charged residues at positions 34 and 39 are critical for mitochondrial targeting. Results suggest that 25% of P4501A1 nascent chains, which escape ER membrane insertion, are processed by a liver cytosolic endoprotease. We postulate that the NH₂-terminal proteolytic cleavage activates a cryptic mitochondrial targeting signal. Immunofluorescence microscopy showed that a portion of transiently expressed P4501A1 is colocalized with the mitochondrial-specific marker protein cytochrome oxidase subunit I. The mitochondrial-associated MT2a and MT2b are localized within the inner membrane compartment, as tested by resistance to limited proteolysis in both intact mitochondria and mitoplasts. Our results therefore describe a novel mechanism whereby proteins with chimeric signal sequence are targeted to the ER as well as to the mitochondria.     

Lack of correlation between binding of Eco RII methyltransferase to DNA duplexes containing mismatches and the promotion of C to T mutations

The cytosine methyltransferases (MTases) M. HhaIand M. HpaII bind substrates in which the target cytosine is replaced by uracil or thymine, i.e. DNA containing a U:G or a T:G mismatch. We have extended this observation to the EcoRII MTase (M. EcoRII) and determined the apparent K_d for binding. Using a genetic assay we have also tested the possibility that MTase binding to U:G mismatches may interfere with repair of the mismatches and promote C:G to T:A mutations. We have compared two mutants of M. EcoRII that are defective for catalysis by the wild-type enzyme for their ability to bind DNA containing U:G or T:G mismatches and for their ability to promote C to T mutations. We find that although all three proteins are able to bind DNAs with mismatches, only the wild-type enzyme promotes C:G to T:A mutations in vivo. Therefore, the ability of M. EcoRII to bind U:G mismatched duplexes is not sufficient for their mutagenic action in cells. Received: 14 November 1996 / Accepted: 17 February 1997     

Simple sequence repeat DNA markers in alfalfa and perennial and annual Medicago species.

Simple sequence repeat (SSR) or microsatellite DNA markers have been shown to function well in plant and mammalian species for genetic map construction and genotype identification. The objectives of the work reported here were to search GenBank for the presence of SSR-containing sequences from the genus Medicago, to assess the presence and frequency of SSR DNA in the alfalfa (Medicago sativa (L.) L. &L.) genome, and to examine the function of selected markers in a spectrum of perennial and annual Medicago species. The screening of an alfalfa genomic DNA library and sequencing of clones putatively containing SSRs indicated approximately 19 000 (AT)n + (CT)n + (CA)n + (ATT)n SSRs in the tetraploid genome. Inheritance was consistent with Mendelian expectations at four selected SSR loci with different core motifs. Additionally, genotypes of a range of Medicago species, including 10 perennial subspecies of the M. sativa complex and other perennial and annual Medicago species, were analyzed at each of the loci to ascertain the presence, number, and size of SSR alleles at each locus in each genotype. These studies indicate that SSR markers can function in alfalfa for the construction of genetic maps and will also be useful in a range of Medicago species for purposes of assessing genetic relatedness and taxonomic relationships, and for genotype identification.     

Interaction of MutS and Vsr: some dominant-negative mutS mutations that disable methyladenine-directed mismatch repair are active in very-short-patch repair

In Escherichia coli and related bacteria, the very-short-patch (VSP) repair pathway uses an endonuclease, Vsr, to correct T-G mismatches that result from the deamination of 5-methylcytosines in DNA to C-G. The products of mutS and mutL, which are required for adenine methylation-directed mismatch repair (MMR), enhance VSP repair. Multicopy plasmids carrying mutS alleles that are dominant negative for MMR were tested for their effects on VSP repair. Some mutS mutations (class I) did not lower VSP repair in a mutS(+) background, and most class I mutations increased VSP repair in mutS cells more than plasmids containing mutS(+). Other plasmid-borne mutS mutations (class II) and mutS(+) decreased VSP repair in the mutS(+) background. Thus, MutS protein lacking functions required for MMR can still participate in VSP repair, and our results are consistent with a model in which MutS binds transiently to the mispair and then translocates away from the mispair to create a specialized structure that enhances the binding of Vsr.     

A gene required for very short patch repair in Escherichia coli is adjacent to the DNA cytosine methylase gene.

Deamination of 5-methylcytosine in DNA results in T/G mismatches. If unrepaired, these mismatches can lead to C-to-T transition mutations. The very short patch (VSP) repair process in Escherichia coli counteracts the mutagenic process by repairing the mismatches in favor of the G-containing strand. Previously we have shown that a plasmid containing an 11-kilobase fragment from the E. coli chromosome can complement a chromosomal mutation defective in both cytosine methylation and VSP repair. We have now mapped the regions essential for the two phenotypes. In the process, we have constructed plasmids that complement the chromosomal mutation for methylation, but not for repair, and vice versa. The genes responsible for these phenotypes have been identified by DNA sequence analysis. The gene essential for cytosine methylation, dcm, is predicted to code for a 473-amino-acid protein and is not required for VSP repair. It is similar to other DNA cytosine methylases and shares extensive sequence similarity with its isoschizomer, EcoRII methylase. The segment of DNA essential for VSP repair contains a gene that should code for a 156-amino-acid protein. This gene, named vsr, is not essential for DNA methylation. Remarkably, the 5' end of this gene appears to overlap the 3' end of dcm. The two genes appear to be transcribed from a common promoter but are in different translational registers. This gene arrangement may assure that Vsr is produced along with Dcm and may minimize the mutagenic effects of cytosine methylation.