Kappa-chain constant-region gene sequences in genus Rattus: coding regions are diverging more rapidly than noncoding regions

We have determined the nucleotide sequence of a 1,200-base pair (bp) genomic fragment that includes the kappa-chain constant-region gene (C kappa) from two species of native Australian rodents, Rattus leucopus cooktownensis and Rattus colletti. Comparison of these sequences with each other and with other rodent C kappa genes shows three surprising features. First, the coding regions are diverging at a rate severalfold higher than that of the nearby noncoding regions. Second, replacement changes within the coding region are accumulating at a rate at least as great as that of silent changes. Third, most of the amino acid replacements are localized in one region of the C kappa domain--namely, the carboxy-terminal "bends" in the alpha-carbon backbone. These three features have previously been described from comparisons of the two allelic forms of C kappa genes in R. norvegicus. These data imply the existence of considerable evolutionary constraints on the noncoding regions (based on as yet undetermined functions) or powerful positive selection to diversify a portion of the constant-region domain (whose physiological significance is not known). These surprising features of C kappa evolution appear to be characteristic only of closely related C kappa genes, since comparison of rodent with human sequences shows the expected greater conservation of coding regions, as well as a predominance of silent nucleotide substitutions within the coding regions.      

Thermodynamic analysis of the folding of the streptococcal protein G IgG-binding domains B1 and B2: why small proteins tend to have high denaturation temperatures.

We have cloned, expressed, and characterized two naturally occurring variations of the IgG-binding domain of streptococcal protein G. The domain is a stable cooperative folding unit of 56 amino acids, which maintains a unique folded structure without disulfide cross-links or tight ligand binding. We have studied the thermodynamics of the unfolding reaction for the two versions of this domain, designated B1 and B2, which differ by six amino acids. They have denaturation temperatures of 87.5 degrees C and 79.4 degrees C, respectively at pH 5.4, as determined by differential scanning calorimetry. Thermodynamic state functions for the unfolding reaction (delta G, delta H, delta S, and delta Cp) have been determined and reveal several interesting insights into the behavior of very small proteins. First, though the B1 domain has a heat denaturation point close to 90 degrees C, it is not unusually stable at physiologically relevant temperatures (delta G = 25 kJ/mol at 37 degrees C). This behavior occurs because the stability profile (delta G vs temperature) is flat and shallow due to the small delta S and delta Cp for unfolding. Related to this point is the second observation that small changes in the free energy of unfolding of the B-domain due to mutation or change in solvent conditions lead to large shifts in the heat denaturation temperature. Third, the magnitude and relative contributions of hydrophobic vs nonhydrophobic forces (per amino acid residue) to the total free energy of folding of the B-domain are remarkably typical of other globular proteins of much larger size.     

NMR structure of HI0004, a putative essential gene product from Haemophilus influenzae, and comparison with the X-ray structure of an Aquifex aeolicus homolog

The solution structure of the 154-residue conserved hypothetical protein HI0004 has been determined using multidimensional heteronuclear NMR spectroscopy. HI0004 has sequence homologs in many organisms ranging from bacteria to humans and is believed to be essential in Haemophilus influenzae, although an exact function has yet to be defined. It has a alpha-beta-alpha sandwich architecture consisting of a central four-stranded beta-sheet with the alpha2-helix packed against one side of the beta-sheet and four alpha-helices (alpha1, alpha3, alpha4, alpha5) on the other side. There is structural homology with the eukaryotic matrix metalloproteases (MMPs), but little sequence similarity except for a conserved region containing three histidines that appears in both the MMPs and throughout the HI0004 family of proteins. The solution structure of HI0004 is compared with the X-ray structure of an Aquifex aeolicus homolog, AQ_1354, which has 36% sequence identity over 148 residues. Despite this level of sequence homology, significant differences exist between the two structures. These differences are described along with possible functional implications of the structures.     

Growth of a Molecular Base for Feeding: The Mind Body Dualism

It is often difficult to identify the ‘who, when, and where’ of advances in medicine and surgery because it's a rare advance indeed (such as the use of digitalis by William Withering) that can be clearly related to the astuteness of one person at one time and place.     

A Hidden Markov Model approach to variation among sites in rate of evolution

The method of Hidden Markov Models is used to allow for unequal and unknown evolutionary rates at different sites in molecular sequences. Rates of evolution at different sites are assumed to be drawn from a set of possible rates, with a finite number of possibilities. The overall likelihood of phylogeny is calculated as a sum of terms, each term being the probability of the data given a particular assignment of rates to sites, times the prior probability of that particular combination of rates. The probabilities of different rate combinations are specified by a stationary Markov chain that assigns rate categories to sites. While there will be a very large number of possible ways of assigning rates to sites, a simple recursive algorithm allows the contributions to the likelihood from all possible combinations of rates to be summed, in a time proportional to the number of different rates at a single site. Thus with three rates, the effort involved is no greater than three times that for a single rate. This "Hidden Markov Model" method allows for rates to differ between sites and for correlations between the rates of neighboring sites. By summing over all possibilities it does not require us to know the rates at individual sites. However, it does not allow for correlation of rates at nonadjacent sites, nor does it allow for a continuous distribution of rates over sites. It is shown how to use the Newton-Raphson method to estimate branch lengths of a phylogeny and to infer from a phylogeny what assignment of rates to sites has the largest posterior probability. An example is given using beta-hemoglobin DNA sequences in eight mammal species; the regions of high and low evolutionary rates are inferred and also the average length of patches of similar rates.      

微卫星跨物种交叉PCR扩增的一个新问题:扩增非同源产物

微卫星已被广泛应用于群体遗传学、生态学和进化生物学研究。然而,一些物种微卫星尚未克隆。为了节省时间和经费,研究人员往往使用一个物种已发表的微卫星引物扩增其近缘物种的微卫星。该研究对属于3个不同科(Clariidae、Heteropneustidae 和Pimelodidae)的7个鲶鱼物种的微卫星跨物种PCR扩增产物进行了序列分析,研究发现扩增非同源（non-orthologous）产物是微卫星跨物种PCR扩增的一个新问题。该研究共采用4对胡子鲶微卫星座位引物对7个鲶鱼物种进行了跨物种PCR扩增。对获得的204个PCR产物的序列分析结果表明,两对微卫星座位引物扩增了所有7个物种的同源特异产物。而其他两个座位的引物扩增了特异但非同源的多态产物,对近缘物种的扩增也获得类似结果。另外,除胡子鲶等位基因大小异源同型(size homoplasy)的特征不明显外,其他物种在3个微卫星座位都具有这一非常明显的特征。这些数据表明,微卫星跨物种间交叉扩增能产生非同源产物;等位基因大小异源同型与微卫星座位本身有关,而与物种间的亲缘关系无明显的相关性。微卫星跨物种扩增产生的非同源产物和等位基因大小异源同型将使系统发育、群体遗传学和进化研究明显复杂化。因此,在应用微卫星跨物种交叉扩增数据以前,最好对跨物种交叉扩增产物进行测序验证。