A single mutation in 16S rRNA that affects mRNA binding and translation-termination.

A single base change in 16S rRNA (C726 to G) has previously been shown to have a dramatic effect on protein synthesis in E. coli (1). This paper more specifically details the effects of the mutation on mRNA binding and translation-termination. The in vitro technique of toeprinting (2) was used to demonstrate that 30S subunits containing the mutation 726G had an altered binding affinity for mRNA by comparison to the wild type. In addition, expression of the mutant ribosomes in vivo resulted in exclusive suppression of the UGA nonsense codon. This effect was supported by in vitro studies that showed the mutant ribosomes to have an altered binding affinity for Release Factor-2.     

Recently amplified Alu family members share a common parental Alu sequence.

Three of the most recently inserted primate Alu family members are exceptionally closely related. Therefore, one, or a few, Alu family members are dominating the amplification process and the vast majority are not actively involved in retroposition. Although individual Alu family members are not under any apparent evolutionary constraint, the sequences of these active members are being moderately conserved.     

Use of novel objects to enhance environments of captive chimpanzees

A small group of chimpanzees was allowed to manipulate different quantities of novel objects in order to enhance their captive conditions. This procedure led to a general increase in the manipulation frequency by the apes. It decreased their inactivity and their self-grooming, and almost eliminated the abnormal behaviors displayed by one individual. The distribution of novel objects did not affect the total frequency of social interactions and did not increase the aggressivity of the animals, even though dominant individuals secured most of the objects when only a small number of them were available. Following their familiarization with the novel objects, the chimpanzees' manipulation frequency decreased whereas self-grooming and abnormal behaviors were increased. Suggestions are made on how to maximize the use of a limited bank of toys to occupy captive chimpanzees.     

Mutagenicity of 2-methylacrolein, 2-ethylacrolein and 2-propylacrolein in Salmonella typhimurium TA100. A comparative study.

The C2-alkylated acrolein derivatives 2-methylacrolein, 2-ethylacrolein and 2-propylacrolein are mutagenic in Salmonella typhimurium TA100. They are direct mutagens, their mutagenic potency being inversely proportional to the size of the alkylating substituent in the C2 position. In the presence of S9 mix, the mutagenicity of all these substances is considerably reduced; the reduction in mutagenicity is inversely proportional to the direct mutagenic potential of the substance. As shown for 2-methylacrolein, the reduction in mutagenicity is dependent on the concentration of S9 in the S9 mix and is not significantly influenced by heat inactivation of the S9 mix or by addition of TCPO, an inhibitor of epoxide hydrolase, to the testing system. There are no indications of enzymatic activation by the metabolizing microsomal system.     

Amplification dynamics of human-specific (HS) Alu family members.

We have investigated the distribution of several recently inserted Alu family members within representatives of diverse human groups. Human population studies using 65 unrelated human DNA samples, as well as a familial study to test inheritance, showed that individual Alu family members could be divided into three groups. The first group consisted of relatively older Alu family members which were monomorphic (homozygous) throughout the population tested (HS C3N1 and C4N6). The second group (HS C4N2, C4N5 and C4N8), apparently inserted into other repetitive regions of the genome, resulting in inconclusive results in the PCR test used. However, it is clear that these particular Alu insertions were present in a majority if not all of the loci tested. The third group was comprised of three dimorphic Alu family members (HS C2N4, C4N4 and TPA 25). Only a single Alu family member (TPA 25) displayed a high degree of dimorphism within the human population. This latter example also showed different allele frequencies in different human groups. The isolation and characterization of additional highly dimorphic Alu family members should provide a useful tool for human population genetics.     

Organization of the micronuclear genome of oxytricha nova

In the hypotrichous ciliated protozoan Oxytricha nova, approximately 95% of the micronuclear genome, including all of the repetitive DNA and most of the unique sequence DNA, is eliminated during the formation of the macronuclear genome. We have examined the interspersion patterns of repetitive and unique and eliminated and retained sequences in the micronuclear genome by characterizing randomly selected clones of micronuclear DNA. Three major classes of clones have been defined: (1) those containing primarily unique, retained sequences; (2) those containing only unique, eliminated sequences; and (3) those containing only repetitive, eliminated sequences. Clones of type one and three document two aspects of organization observed previously: clustering of macronuclear destined sequences and the presence of a prevalent repetitive element. Clones of the second type demonstrate for the first time that eliminated unique sequence DNA occurs in long stretches uninterrupted by repetitive sequences. To further examine repetitive sequence interspersion, we characterized the repetitive sequence family that is present in 50% of the clones (class three above). A consensus map of this element was obtained by mapping approximately 80 phage clones and by hybridization to digests of micronuclear DNA. The repeat element is extremely large (approximately 24 kb) and is interspersed with both macronuclear destined sequences and eliminated unique sequences.     

Modified activity of Aeromonas aminopeptidase: metal ion substitutions and role of substrates

Aeromonas aminopeptidase contains two nonidentical metal binding sites that have been shown by both spectroscopy and kinetics to be capable of interacting with one another [Prescott, J.M., Wagner, F.W., Holmquist, B., & Vallee, B.L. (1985) Biochemistry 24, 5350-5356]. The effects of metal ion substitutions on the susceptibility of the p-nitroanilides of L-alanine, L-valine, and L-leucine--substrates that are hydrolyzed at widely differing rates by native Aeromonas aminopeptidase--were studied by determining values of kcat and Km for the 16 metalloenzymes that result from all possible combinations of Zn2+, Co2+, Ni2+, and Cu2+ in each of the two sites. The different combinations of metal ions and substrates yield a broad range in kinetic values; kcat varies by more than 1800-fold, Km by 3000-fold, and kcat/Km ratios by more than 10,000. L-Leucine-p-nitroanilide is by far the most susceptible of the three substrates, and the hyperactivation previously observed with aminopeptidase containing either Ni2+ or Cu2+ in the first binding site and Zn2+ in the second site occurs only with the two poorer substrates, L-alanine-p-nitroanilide and L-valine-p-nitroanilide. Although the enzyme with Zn2+ in both sites hydrolyzes the substrates with N-terminal alanine and valine poorly, it is extremely effective toward L-leucine-p-nitroanilide. Neither metal binding site can be identified as controlling either Km or kcat; both parameters are influenced by the identity of the metal ions, by the site each occupies, and, most strongly, by the substrate.(ABSTRACT TRUNCATED AT 250 WORDS)     

The slow, tight binding of bestatin and amastatin to aminopeptidases

Bestatin reversibly inhibits Aeromonas aminopeptidase (EC 3.4.11.10) in a process that is remarkable for its unusual degree of time dependence. The binding of bestatin by both Aeromonas aminopeptidase and cytosolic leucine aminopeptidase (EC 3.4.11.1) is slow and tight, with Ki values (determined from rate constants) of 1.8 X 10(-8) and 5.8 X 10(-10) M, respectively. In contrast, microsomal aminopeptidase (EC 3.4.11.2) binds bestatin in a rapidly reversible process with a Ki value of 1.4 X 10(-6) M. Kinetic analysis of the slow inhibition observed is facilitated by the use of a variety of experimental treatments, primarily measurements made during pre-equilibrium; however, careful selection of conditions permits use also of steady state observations. When titrated with bestatin, 1 mol of cytosolic leucine aminopeptidase (containing 6 g atoms each of zinc and manganese) is rendered 80% inactive by 1 mol of inhibitor, thus suggesting that enzymatic activity depends on one active site/hexamer; titration of Aeromonas aminopeptidase by bestatin reveals a 1:1 stoichiometry. Amastatin inhibits all three aminopeptidases through the mechanism of slow, tight binding with Ki values ranging from 3.0 X 10(-8) to 2.5 X 10(-10) M. This behavior of microsomal aminopeptidase contrasts sharply with its rapidly reversible inhibition by bestatin. The slow, tight binding observed with five of the six aminopeptidase-inhibitor pairs investigated suggests the formation of a transition state analog complex between the enzyme and inhibitor. Physical evidence consistent with this possibility was provided by the observation that both bestatin and amastatin perturb the absorption spectrum of cobalt Aeromonas aminopeptidase.     

Integration site preferences of the Alu family and similar repetitive DNA sequences.

Numerous flanking nucleotide sequences from two primate interspersed repetitive DNA families have been aligned to determine the integration site preferences of each repetitive family. This analysis indicates that both the human Alu and galago Monomer families were preferentially inserted into short d(A+T)-rich regions. Moreover, both primate repeat families demonstrated an orientation specific integration with respect to dA-rich sequences within the flanking direct repeats. These observations suggest that a common mechanism exists for the insertion of many repetitive DNA families into new genomic sites. A modified mechanism for site-specific integration of primate repetitive DNA sequences is provided which requires insertion into dA-rich sequences in the genome. This model is consistent with the observed relationship between galago Type II subfamilies suggesting that they have arisen not by mere mutation but by independent integration events.