Replacement Substitution Research Articles

Evolutionary relationship and secondary structure predictions in four transport proteins of Saccharomyces cerevisiae.

The comparison of the amino acid sequences of four yeast transport proteins indicates that there is a questionable relatedness between the uracil permease (FUR4) and the purine-cytosine permease (FCY2), whereas the arginine permease (CAN1) and the histidine permease (HIP1) clearly originated from a common molecular ancestor. The analysis of the primary structure of these transport proteins by two methods of secondary structure predictions suggests the presence of 9-12 membrane-spanning alpha-helices in each polypeptide chain. These results are concordant in that 90% of the alpha-helices were determined by both methods to be at the same positions. In the aligned sequences HIP1 and CAN1, the postulated membrane-spanning alpha-helices often start at corresponding sites, even though the overall sequence similarity of the two proteins is only 30%. In the aligned DNA coding sequences of CAN1 and HIP1, synonymous nucleotide substitutions occur with very similar frequencies in regions where the replacement substitution (changing the amino acids) frequencies are widely different. Moreover, our data suggest that the replacement substitutions can be considered as neutral in the N-terminal segment, whereas the other regions are subject to a conservative selective pressure because, if compared to a random drift, the replacement substitutions are underrepresented.

Recent origin for a thermostable alcohol dehydrogenase allele of Drosophila melanogaster.

The nucleotide sequence of the Fast-Chateau Douglas isolate of the thermostable alcohol dehydrogenase allele is compared with the sequences of the Slow and Fast alleles of Drosophila melanogaster. Conceptual translation of the FChD sequence indicates that the thermostable polypeptide has the diagnostic FAST amino acid replacement at residue 192 and an additional replacement of serine for proline at residue 214. This suggests a Fast origin for the thermostable Adh allele. However, some of the biochemical properties of the FCHD protein resemble those of the SLOW rather than the FAST polypeptides. The serine for proline replacement confers upon the thermostable polypeptide substrate specificities and some kinetic parameters similar to the SLOW protein. The same replacement substitution within the third coding exon also appears to alter the ADH protein concentration to a level similar to the SLOW polypeptide and the probable effect is at the level of mRNA concentration. The low level of nucleotide sequence variation, other than that leading to the amino acid substitution, suggests a recent origin for the thermostable allele. The time since divergence of the FChD sequence from Fast is estimated to be approximately 260,000-470,000 years.

Sequence analysis of cDNAs for the human and bovine ATP synthase ? subunit: mitochondrial DNA genes sustain seventeen times more mutations

We have cloned and sequenced human and bovine cDNAs for the beta subunit of the ATP synthase (ATP-syn beta), a nuclear DNA (nDNA) encoded oxidative phosphorylation (OXPHOS) gene. The two cDNAs were found to share 99% amino acid homology and 94% nucleotide homology. The evolutionary rate of ATPsyn beta was then compared with that of two mitochondrial DNA (mtDNA) ATP synthase genes (ATPase 6 and 8), seven other mtDNA OXPHOS genes, and a number of nuclear genes. The synonymous substitution rate for ATPsyn beta proved to be 1.9 x 10(-9) substitutions per site per year (substitutions x site-1 x year-1) (SSY). This is less than 1/2 that of the average nDNA gene, 1/12 the rate of ATPase 6 and 8, and 1/17 the rate of the average mtDNA gene. The synonymous and replacement substitution rates were used to calculate a new parameter, the "selective constraint ratio". This revealed that even the most variable mtDNA protein was more constrained than the average nDNA protein. Thus, the high substitution mutation rate and strong selective constraints of mammalian mtDNA proteins suggest that mtDNA mutations may result in a disproportionately large number of human hereditary diseases of OXPHOS.

Use of P-Element-Mediated Transformation to Identify the Molecular Basis of Naturally Occurring Variants Affecting Adh Expression in Drosophila melanogaster

The purpose of the work reported here is to identify the molecular basis of the difference in level of expression between the polymorphic Slow and Fast alcohol dehydrogenase (Adh) alleles in Drosophila melanogaster. Previous studies have shown that Fast lines typically have a two- to threefold higher activity level than Slow lines and they also have a substantially higher level of ADH-protein (estimated immunologically). The results of a restriction fragment length polymorphism study in relation to ADH activity variation had previously suggested that the difference in Adh expression between allozymes might not be due entirely to the amino acid replacement substitution, but could be due in part to linkage disequilibrium with a regulatory site polymorphism. Here we describe an approach that makes use of P-element-mediated transformation in order to identify the nucleotide substitution(s) responsible for this difference in ADH level. This approach consists of generating recombinants in vitro between Adh region clones derived from a typical Slow/Fast pair of alleles and then testing for the effects of particular restriction fragments on expression in vivo by transformation. Using this approach, the effect on both ADH activity and ADH-protein level clearly maps to a 2.3-kb restriction fragment that includes all of the Adh coding sequence and some intron and 3' flanking sequence, but excludes all of the 5' flanking sequence of the distal (adult) transcriptional unit. Comparison of Kreitman's DNA sequences for this fragment from several Slow and Fast alleles showing the typical difference in activity level shows that only three nucleotide substitutions distinguish all Fast from all Slow alleles. Thus, it is likely that one or more of these substitutions causes the major difference in Adh expression between allozymic classes. One of these substitutions is, of course, the Slow/Fast amino acid replacement substitution (at 1490) while the other two are nearby third position silent substitutions (at 1443 and 1527). A quantitative analysis of variation among transformant stocks shows that the P-element transformation approach can be used to localize even relatively small effects on gene expression (on the order of 20%).

VH genes encoding the immune response to beta-(1,6)-galactan: somatic mutation in IgM molecules.

The immune response to beta-(1,6)-galactan in the BALB/c mouse has been well characterized and includes the amino acid sequence determination of 13 monoclonal antibodies. The genetic potential encoding the VH regions of these antibodies has been determined by isolation and sequencing of homologous germline genes. The germline repertoire encoding these proteins was found to consist of two closely related genes. One of these directly encodes the VH segments of seven Gal-binding proteins, and the second directly encodes one additional protein sequence. Sequence variations found in the VH regions of five other Gal-binding proteins can be explained by somatic mutations leading to single base substitutions in the more frequently used gene. Since four of the hybridoma proteins exhibiting somatic mutations are of the IgM class, these results indicate that somatic mutation, in this system, is not associated with class switching and can apparently be initiated early in B-cell development. The two Gal genes are the only members of a very restricted multigene family and probably result from a gene duplication estimated to occur 1.4-2.8 million years ago. Three other genes hybridizing at moderate stringency to a VHGal probe were also sequenced and were found to be members of two additional VHIII families. Studies of the silent to replacement substitution ratios of these and other VH genes indicate that the number of silent substitutions found in immunoglobulin VH genes is lower than expected when compared with proteins such as preproinsulin and globin. Analysis of base composition reflected in these sequences indicates a marked increase of A-T% in the first and second codon positions of complementarity determining regions (CDR) which may be important in facilitating point mutations.

Some ring replacement and nucleophilic substitution reactions of η 6-subsituted arene-η 5-cylopentadienyliron hexafluorophosphates

A number of nitroarene and aminoarene complexes, including the PF 6 − salts of NO 2C 6H 5FeCp p+, 0-, m- or p-CH 3(NO 2)C 6H 4FeCp +, NH 2C 6H 5FeCp p+ and 0-, m-, or p-CH 3(NH 2)C 6H 4FeCp p+, when heated with an excess of P(OC 2H 5) 3 all gave rise to the ring replacement product, CpFe(P(OC 2H 5) 3) 3 + PF 6 − (I). Similarly, the thermal reaction of NO 2C 6H 5Fe(CH 3)Cp + PF 6 − or NH 2C 6H 5Fe(CH 3)Cp + PF 6 − with P(OC 2H 5) 3 gave CH 3CpFe(P(OC 2H 5) 3) 3 + PF 6 − (VII). With m-CH 3(Cl)C 6H 4-FeCp + PF 6 − (XIV), heating with P(OC 2H 5) 3 also gave rise to I, while the same treatment with P(OC 2H 5) 3 at room temperature in CH 2Cl 2 showed no nucleophilic substitution of the chlorine atom of XIV by P(OC 2H 5) 3. On the other hand, the chlorine atom of a number of chloroarene complexes could be readily displaced at room temperature with various amines acting as nucleophiles. Such nucleophilic substitutions were carried out on ClC 6H 5FeCp + PF 6 − and 0-, m- or p-CH 3(CI)C 6H 4FeCp + PF 6 − with methylamine, ethylenediamine, cyclohexylamine, benzylamine and pyrrolidine to give rise to 20 N-substituted aminoarene complexes.

Physical Classification of Carbonate Cement in Quartzose Sandstones

ABSTRACT The proposed classification of carbonate cement precipitated in quartzose sandstones is based upon two major characteristics: (1) crystallization of carbonate in available intergranular pores without modification in the arrangement of the detrital grain framework, or in the shape of such grains; (2) crystallization of cement involving expansion of the intergranular space and consequent rearrangement in the packing of detrital grains; or, crystallization of cement involving replacement and mineral substitution of detrital grains. The simple cement fills pores only and serves as a lithifying agent to preserve the fabric of the uncemented sediment. Cementation which is accompanied by volume expansion modifies grain-supported fabrics, can cause rupture of porous grains such as glauconite, can crystallize as concretions around grains, or as large, single poikilitic crystals, can replace grains of silicate minerals, or modify an original framework of carbonate grains by recrystallization.

Re-Vocalized Films

THE replacement of voices on talking picture films may be accomplished in a relatively simple manner by an invention which for purposes of patent title has been termed a Vivigraph. A film in which voice replacement or voice substitution has been effected may conveniently be termed a Re-Vocalized Film.