Rates of nuclear and cytoplasmic mitochondrial DNA sequence divergence in mammals

Differential rates of nucleotide substitution among different gene segments and between distinct evolutionary lineages is well documented among mitochondrial genes and is likely a consequence of locus-specific selective constraints that delimit mutational divergence over evolutionary time. We compared sequence variation of 18 homologous loci (15 coding genes and 3 parts of the control region) among 10 mammalian mitochondrial DNA genomes which allowed us to describe different mitochondrial evolutionary patterns and to produce an estimation of the relative order of gene divergence. The relative rates of divergence of mitochondrial DNA genes in the family Felidae were estimated by comparing their divergence from homologous counterpart genes included in nuclear mitochondrial DNA (Numt, pronounced "new might"), a genomic fossil that represents an ancient transfer of 7.9 kb of mitochondrial DNA to the nuclear genome of an ancestral species of the domestic cat (Felis catus). Phylogenetic analyses of mitochondrial (mtDNA) sequences with multiple outgroup species were conducted to date the ancestral node common to the Numt and the cytoplasmic (Cymt) mtDNA genes and to calibrate the rate of sequence divergence of mitochondrial genes relative to nuclear homologous counterparts. By setting the fastest substitution rate as strictly mutational, an empirical "selective retardation index" is computed to quantify the sum of all constraints, selective and otherwise, that limit sequence divergence of mitochondrial gene sequences over time.      

Slow oscillations as a probe of the dynamics of the locus coeruleus-frontal cortex interaction in anesthetized rats

Multiunit or single unit activity recorded simultaneously from frontal cortex (FC) and locus coeruleus (LC) under ketamine anesthesia revealed that both regions show slow oscillatory activity, together or separately. If, however, both regions are engaged in this oscillatory activity, there is a systematic relationship between their phases with peak LC firing always following FC firing by 200–400 ms. This was confirmed by cross-correlational analyses, which indicated that the two structures temporarily form a resonant system. The FC-LC resonant state is, however, loose enough to remain open to other intrinsic or extrinsic influences, keeping the measured frequencies of oscillations at each site slightly different, as demonstrated by a delailed analysis of the autocorrelograms. An injection of lidocaine at the frontal cortex site, while sharply reducing the prefrontal activity to essentially zero, leads to an increase of the LC activity and to a modification of the shape of the LC autocorrelogram, but does not change appreciably the phase relationship between the activity in the two structures during the diminishing activity in FC.     

Sedimentation equilibrium measurements of the intermediate-size tobacco mosaic virus protein polymers

Short-column sedimentation equilibrium methods have been applied for the first time to tobacco mosaic virus (TMV) protein (0.1 M ionic strength orthophosphate) at pH 6.5 and at pH 7.0 to estimate molecular weights. Previous sedimentation velocity experiments at pH 6.5, 20 degrees C have led to the conclusion that the major boundary with an S0(20),w value of 24.4 +/- 0.1 S consists of a distribution of polymers which are mainly three-turn, 48-51-subunit helical rod aggregates. The directly measured z-average molecular weights together with sedimentation velocity data are entirely consistent with this assignment of a three-turn aggregate. Molecular weights have also been determined under two conditions where a large mass fraction of the protein sediments with an S0(20),w value of 20.3 +/- 0.2 S. At pH 6.5, 6-8 degrees C, the aggregates in this boundary are metastable and correspond to 50-60% of the preparation. At pH 7.0, 20 degrees C at equilibrium, 65-75% of the protein sediments at 20.3 S. The 20.3S boundary is very similar under both conditions and is interpreted as being composed of a distribution of protein aggregates centered about 39 +/- 2 subunits. This result is important in the interpretation of previous kinetic measurements of TMV self-assembly. The current view is that the 34-subunit structure of TMV protein, in the form of a cylindrical disk which is made up of two 17-subunit layers and has been characterized in single-crystal X-ray diffraction studies, plays a central role in the initial binding steps with RNA. The present results are not consistent with the view that there is a significant concentration of the TMV protein disk structure in solution under the usual conditions of TMV self-assembly.     

Cloning and expression in Escherichia coli of the gene encoding Aspergillus flavus urate oxidase.

Amino acid sequencing of peptides obtained after proteolytic hydrolysis of Aspergillus flavus urate oxidase (uricase) permitted the design of oligodeoxynucleotide probes that were used to obtain 1.2- and 5-kilobase pair DNA fragments from A. flavus cDNA and genomic libraries, respectively. The cDNA fragment contained the entire coding region for uricase, and comparison with the genomic fragment revealed the presence of two short introns in the coding region of the gene. A. flavus uricase has around 40% overall identity with uricases from higher organisms but with many conserved amino acids. Hitherto highly conserved consensus patterns found in other uricases were found to be modified in the A. flavus enzyme, notably the sequence Val-Leu-Lys-Thr-Thr-Gln-Ser near position 150, which in the filamentous fungus is uniquely modified to Val-Leu-Lys-Ser-Thr-Asn-Ser. Silent mutations were introduced by cassette mutagenesis near the 5'-extremity of the coding sequence in order to conform with Escherichia coli codon usage, and the uricase was expressed in the E. coli cytoplasm in a completely soluble, biologically active form.     

Self-association of human and porcine relaxin as assessed by analytical ultracentrifugation and circular dichroism.

The self-association properties of recombinant DNA derived human relaxin, and porcine relaxin isolated from porcine ovaries, have been studied by sedimentation equilibrium analytical ultracentrifugation and circular dichroism (CD). The human relaxin ultracentrifuge data were adequately defined by a monomer-dimer self-association model with an association constant of approximately 6 x 10(5) M-1, whereas porcine relaxin was essentially monomeric in solution. An approximate 5-fold increase in weight fraction of human relaxin monomer elicited by dilution of the protein resulted in no change in the far-UV CD spectrum at 220 nm. In contrast, after the same increase in weight fraction of monomer, the near-UV circular dichroism spectra for human relaxin exhibited a significant decrease in the amplitude for the CD bands near 277 and 284 nm. These CD bands, which may be assigned to the lone tyrosine in human relaxin, are superimposed on a broad envelope that is probably due to the three disulfide chromophores. Although both the human and porcine proteins contain two tryptophan residues, the near-UV CD spectra exhibit only a broad shoulder near 295 nm rather than the strong CD bands often found for tryptophan. Moreover, there is little change in this broad band after dilution of human relaxin to concentrations that resulted in a 4-fold increase in monomer weight fraction. These data suggest that dissociation of the human relaxin dimer to monomer is not accompanied by large overall changes in secondary structure or alteration in the average tryptophan environment, whereas there is a significant change in the tyrosine environment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple actins in drosophila melanogaster

The tissue and developmental specificities of the three Drosophila isoactins, originally identified in primary myogenic cultures and in the permanent Schneider L-2 cell line, have been investigated. Of these three isoactins (I, II, and III), actins I and II are stable and actin III is unstable. Two-dimensional polyacrylamide gel electrophoretic analyses of total cellular extracts after 1-h [(35)S]methionine pulses were performed on a large variety of embryonic, larval, and adult muscle and nonmuscle tissues. The results suggest that isoactins II and III are generalized cellular actins found in all drosophila cell types. Actin I, on the other hand, is muscle-associated and is found exclusively in supercontractile muscle (such as larval body wall and larval and adult viscera) including primary myogenic cell cultures. Although actin I synthesis is not detectable during very early embryogenesis, it is detectable by 25 h and actin I is a major stable actin in all larval muscle tissues. Actin I is synthesized in reduced amounts relative to the other actins in late third instar larvae but is again a major product of actin synthesis in the adult abdomen. A stable actin species with the same pI as actin III has been identified in the adult thorax and appears to be unique to flight muscle tissue. This new stable form of thoracic actin may be the result of a stabilization of the actin III found in other tissues or may be an entirely separate gene product.     

Selective inhibition of prostaglandin biosynthesis by gold salts and phenylbutazone

Gold salts and phenylbutazone selectively inhibit the synthesis of PGF_2α and PGE₂ respectively. Lowered production of one prostaglandin species is accompanied by an increased production of the other. Selective inhibition by these drugs was observed in the presence of adrenaline, reduced glutathione and copper sulphate under conditions when most anti-inflammatory compounds inhibited PGE₂ and PGF_2α syntheses equally. It is postulated that selective inhibitors may have a different mode of action in vivo and beneficial effects may be related to the endogenous ratio of PGE to PGF required for normal function.     

Formation of temporary flagellar structures during insect organogenesis

Cilia and flagella are rare in nongerminal tissues of anthropods, and are generally thought to be restricted to sperm and sensory cells in insects (2). Whitten (5) has reported the presence of kinetosomes at the base of mitotrichia in the dipteran fly Sarcophaga bullata, but reports no evidence of the organization of fibrous elements characteristic of cilia and or flagella. During an ultrastructural analysis of morphogenesis of the colleterial gland of the silk moth Hyalophora cecropia, we found the first example of paired flagella associated with an insect secretory cell. These structures are also unusual in that they serve a temporary role in morphogenesis and subsequently disappear at the terminal stages of differentiation.