Four-body potentials reveal protein-specific correlations to stability changes caused by hydrophobic core mutations.

Mutational experiments show how changes in the hydrophobic cores of proteins affect their stabilities. Here, we estimate these effects computationally, using four-body likelihood potentials obtained by simplicial neighborhood analysis of protein packing (SNAPP). In this procedure, the volume of a known protein structure is tiled with tetrahedra having the center of mass of one amino acid side-chain at each vertex. Log-likelihoods are computed for the 8855 possible tetrahedra with equivalent compositions from structural databases and amino acid frequencies. The sum of these four-body potentials for tetrahedra present in a given protein yields the SNAPP score. Mutations change this sum by changing the compositions of tetrahedra containing the mutated residue and their related potentials. Linear correlation coefficients between experimental mutational stability changes, Delta(DeltaG(unfold)), and those based on SNAPP scoring range from 0.70 to 0.94 for hydrophobic core mutations in five different proteins. Accurate predictions for the effects of hydrophobic core mutations can therefore be obtained by virtual mutagenesis, based on changes to the total SNAPP likelihood potential. Significantly, slopes of the relation between Delta(DeltaG(unfold)) and DeltaSNAPP for different proteins are statistically distinct, and we show that these protein-specific effects can be estimated using the average SNAPP score per residue, which is readily derived from the analysis itself. This result enhances the predictive value of statistical potentials and supports previous suggestions that "comparable" mutations in different proteins may lead to different Delta(DeltaG(unfold)) values because of differences in their flexibility and/or conformational entropy.

Importance of quantitative genetic variations in the etiology of hypertension.

Recent progress has been remarkable in identifying mutations which cause diseases (mostly uncommon) that are inherited simply. Unfortunately, the common diseases of humankind with a strong genetic component, such as those affecting cardiovascular function, have proved less tractable. Their etiology is complex with substantial environmental components and strong indications that multiple genes are implicated. In this article, we consider the genetic etiology of essential hypertension. After presenting the distribution of blood pressures in the population, we propose the hypothesis that essential hypertension is the consequence of different combinations of genetic variations that are individually of little consequence. The candidate gene approach to finding relevant genes is exemplified by studies that identified potentially causative variations associated with quantitative differences in the expression of the angiotensinogen gene (AGT). Experiments to test causation directly are possible in mice, and we describe their use to establish that blood pressures are indeed altered by genetic changes in AGT expression. Tests of differences in expression of the genes coding for the angiotensin-converting enzyme (ACE) and for the natriuretic peptide receptor A are also considered, and we provide a tabulation of all comparable experiments in mice. Computer simulations are presented that resolve the paradoxical finding that while ACE inhibitors are effective, genetic variations in the expression of the ACE gene do not affect blood pressure. We emphasize the usefulness of studying animals heterozygous for an inactivating mutation and a wild-type allele, and briefly discuss a way of establishing causative links between complex phenotypes and single nucleotide polymorphisms.

Patterned library analysis: a method for the quantitative assessment of hypotheses concerning the determinants of protein structure.

Site-directed mutagenesis and combinatorial libraries are powerful tools for providing information about the relationship between protein sequence and structure. Here we report two extensions that expand the utility of combinatorial mutagenesis for the quantitative assessment of hypotheses about the determinants of protein structure. First, we show that resin-splitting technology, which allows the construction of arbitrarily complex libraries of degenerate oligonucleotides, can be used to construct more complex protein libraries for hypothesis testing than can be constructed from oligonucleotides limited to degenerate codons. Second, using eglin c as a model protein, we show that regression analysis of activity scores from library data can be used to assess the relative contributions to the specific activity of the amino acids that were varied in the library. The regression parameters derived from the analysis of a 455-member sample from a library wherein four solvent-exposed sites in an alpha-helix can contain any of nine different amino acids are highly correlated (P < 0.0001, R(2) = 0. 97) to the relative helix propensities for those amino acids, as estimated by a variety of biophysical and computational techniques.

Nonideality and protein thermal denaturation.

We studied the thermal denaturation of eglin c by using CD spectropolarimetry and differential scanning calorimetry (DSC). At low protein concentrations, denaturation is consistent with the classical two-state model. At concentrations greater than several hundred microM, however, the calorimetric enthalpy and the midpoint transition temperature increase with increasing protein concentration. These observations suggested the presence of intermediates and/or native state aggregation. However, the transitions are symmetric, suggesting that intermediates are absent, the DSC data do not fit models that include aggregation, and analytical ultracentrifugation (AUC) data show that native eglin c is monomeric. Instead, the AUC data show that eglin c solutions are nonideal. Analysis of the AUC data gives a second virial coefficient that is close to values calculated from theory and the DSC data are consistent with the behavior expected for nonideal solutions. We conclude that the concentration dependence is caused by differential nonideality of the native and denatured states. The nondeality arises from the high charge of the protein at acid pH and is exacerbated by low buffer concentrations. Our conclusion may explain differences between van't Hoff and calorimetric denaturation enthalpies observed for other proteins whose behavior is otherwise consistent with the classical two-state model.

Contaminated casualties: are we prepared to receive them?

The NHS's reception of casualties contaminated by radiation is reviewed. The findings suggest that training, facilities and personal protection for hospital staff are inadequate.

Rodent L1 evolution has been driven by a single dominant lineage that has repeatedly acquired new transcriptional regulatory sequences.

All mammalian genomes contain approximately 100,000 copies of the transposable element LINES-1 (L1). Phylogenetic analysis indicates that the L1 progenitor predates the mammalian radiation; since that time, the open reading frames encoded in L1 have evolved under selection. The least conserved regions within L1 are the 5'-terminal transcriptional regulatory sequences. In rodents, four types of L1 elements (A, F, and V from mouse and R from rat) have been defined according to the type of apparently nonhomologous promoter sequence present at the 5' end. In this study, we investigate the relationships between these four types of promoters. DNA sequence was determined from approximately 1.5-kb regions from the 5' ends of seven F- and three V-type L1 elements. These sequences were aligned with 29 previously reported L1 elements. Phylogenetic analysis was then performed on the homologous regions of the alignment. The results indicate that in mouse all of the A-, F-, and V-type elements belong to a single dominant lineage but were inserted into the genome during different time periods; V-type elements are the oldest, while A-type elements are the most recently inserted. V-type elements also appear ancestral to the R-type elements found in rat and therefore were replicatively competent prior to the divergence of rat and mouse. Analysis of sequence identity indicates that the different 5' promoters did not derive from a common ancestor. Therefore, the dominant L1 lineage appears to have acquired novel promoter sequences from non-L1 sources. Transposable elements from a wide range of species show similar structural rearrangements, suggesting that acquisition of new sequences may be a common theme in their evolution.

Molecular resurrection of an extinct ancestral promoter for mouse L1.

The F-type subfamily of LINE-1 or L1 retroposons [for long interspersed (repetitive) element 1] was dispersed in the mouse genome several million years ago. This subfamily appears to be both transcriptionally and transpositionally inactive today and therefore may be considered evolutionarily extinct. We hypothesized that these F-type L1s are inactive because of the accumulation of mutations. To test this idea we used phylogenetic analysis to deduce the sequence of a transpositionally active ancestral F-type promoter, resurrected it by chemical synthesis, and showed that it has promoter activity. In contrast, F-type sequences isolated from the modern genome are inactive. This approach, in which the automated DNA synthesizer is used as a "time machine," should have broad application in testing models derived from evolutionary studies.

A major difference between the divergence patterns within the lines-1 families in mice and voles.

L1 retroposons are represented in mice by subfamilies of interspersed sequences of varied abundance. Previous analyses have indicated that subfamilies are generated by duplicative transposition of a small number of members of the L1 family, the progeny of which then become a major component of the murine L1 population, and are not due to any active processes generating homology within preexisting groups of elements in a particular species. In mice, more than a third of the L1 elements belong to a clade that became active approximately 5 Mya and whose elements are > or = 95% identical. We have collected sequence information from 13 L1 elements isolated from two species of voles (Rodentia: Microtinae: Microtus and Arvicola) and have found that divergence within the vole L1 population is quite different from that in mice, in that there is no abundant subfamily of homologous elements. Individual L1 elements from voles are very divergent from one another and belong to a clade that began a period of elevated duplicative transposition approximately 13 Mya. Sequence analyses of portions of these divergent L1 elements (approximately 250 bp each) gave no evidence for concerted evolution having acted on the vole L1 elements since the split of the two vole lineages approximately 3.5 Mya; that is, the observed interspecific divergence (6.7%-24.7%) is not larger than the intraspecific divergence (7.9%-27.2%), and phylogenetic analyses showed no clustering into Arvicola and Microtus clades.

L1 A-monomer tandem arrays have expanded during the course of mouse L1 evolution.

LINE-1 (L1) is a family of highly repeated DNA sequences interspersed throughout the mammalian genome. Individual L1 elements are thought to be generated by a transposition mechanism involving reverse transcription of an RNA intermediate followed by insertion into a new genomic site. In mice, three major families of L1 elements, termed "A," "F," and "V," have been defined on the basis of the sequence found at the 5' terminus. Previous analyses of A-monomers have demonstrated sequence heterogeneity among individual A-monomers, variation in the length of A-monomer sequences, and the presence of transcriptional regulatory activity. To provide a detailed characterization of A-monomers as a foundation for studying their transcriptional regulatory activity, we have analyzed the sequences of 39 complete or partial length A-monomers from 20 different mouse L1 elements. A-monomers can be classified into six different types according to shared-sequence length variations. Consensus sequences for the six types of A-monomers indicate conservation of possible transcription factor-binding sequences. Specific subgroups of A-monomers correlate with the relative dispersal time of a mouse L1 element. A phylogenetic analysis of A-monomers indicates that the length variants represent good diagnostic sites for phylogenetic subgroups of A-monomer sequences. These observations suggest a model for the evolution of A-monomer tandem arrays that involves stepwise mutation and array expansion in the 5' direction. Hybridization data provide a minimum estimate of 16,000 copies of the A-monomer sequence in the mouse haploid genome, with an average array length of 2.1 monomer units.

Master genes in mammalian repetitive DNA amplification.

The analysis of species-specific subfamilies of both the LINE and SINE mammalian repetitive DNA families suggests that such subfamilies have arisen by amplification of an extremely small group of 'master' genes. In contrast to the master genes, the vast majority of both SINEs and LINEs appear to behave like psudogenes in their inability to undergo extensive amplification.

Reduction of fibrinogen adsorption on PEG-coated polystyrene surfaces.

Reduction of protein adsorption by coating surfaces with polyethylene glycol (PEG) is well documented. The present work has four goals related to these previous studies: first, to develop chemistry providing densely packed, covalently bound PEG on polystyrene (PS); second, to determine the ability of these modified surfaces to reject fibrinogen; third, to compare the protein-rejecting ability of branched and linear PEGs; and fourth, to examine the utility of an ELISA-type procedure for measuring protein adsorption. It was found that PEG-epoxide could be readily coupled to amine groups of poly(ethylene imine) (PEI), which had been preadsorbed onto an oxidized PS surface. The PEG groups on branched PEGs appear to act as an excluded volume to repel proteins, similar to arguments previously raised for linear PEGs. The results of protein adsorption studies showed that fibrinogen adsorption is significantly reduced by coating polystyrene with either linear or branched PEGs of 1500 to 20,000 in molecular weight. The ELISA technique was found to be equivalent in sensitivity to radiolabeled fibrinogen for estimating adsorption levels. It is expected that PEG-coated PS will have much utility in a variety of biomedical applications.

Strand-specific LINE-1 transcription in mouse F9 cells originates from the youngest phylogenetic subgroup of LINE-1 elements.

LINE-1 (L1) is a mammalian family of highly repeated DNA sequences that are members of a class of transposable elements whose movement involves an RNA intermediate. Both structural and evolutionary data indicate that the L1 family consists of a small number of active transposable elements interspersed with a large number of L1 pseudogenes. In the mouse, the longest, characterized L1 sequences span about 7000 base-pairs and contain two long open reading frames. Two subfamilies of mouse L1 elements, A and F, have been defined on the basis of the type of putative transcriptional regulatory sequence found at the 5' end. In order to identify a transcribed subset of L1 elements in mouse F9 teratocarcinoma cells, we have examined the strand-specificity of L1 transcription by Northern analysis and compared the open reading frame-1 sequences of ten A-type cDNAs with fifteen genomic A-type L1 elements. Transcripts containing A-type sequence are far more abundant than those containing F-type sequence. Although the majority of L1 RNA in F9 cells appears to be transcribed non-specifically from both strands, our results provide evidence for a subpopulation of variable length, strand-specific transcripts arising from A-type transcriptional regulatory sequences. F9 cell cDNA sequences, which share greater than 99.5% sequence identity with one another, represent a homogeneous subset of the genomic L1 population. Examination of genomic mouse L1 sequences reveals three types of length polymorphism in a defined segment of the first open reading frame. Phylogenetic analysis shows a correlation between the type of length polymorphism in the first open reading frame and the relative age of an individual A-type genomic L1 element. Comparison of the cDNA and genomic sequences indicates that the youngest subgroup of A-type L1 elements is preferentially transcribed in F9 cells. This subgroup may be currently dominating the L1 dispersal process in mice.

Identification of transcriptional regulatory activity within the 5' A-type monomer sequence of the mouse LINE-1 retroposon.

LINE-1 (L1) is a retroposon found in all mammals. In the mouse, approximately 10% of L1 elements are full-length and can be grouped into two classes, A or F, based upon the type of monomer sequence repeated at the 5' end. In order to test for promoter activity in the 5' end of the A-type mouse L1 element, we cloned several different A-monomers into a promoterless chloramphenicol acetyltransferase (CAT) vector. The A-monomer constructs varied in their ability to regulate transcription of the CAT gene, exhibiting CAT activity 16-37% of that detected with the Rous sarcoma virus promoter and enhancer. A series of A-monomer deletions were tested for their ability to regulate CAT expression and gel retardation experiments were performed to identify regions of the A-monomer that may be involved in L1 transcriptional regulation. A-monomer sequences are usually found repeated 2-5 times at the 5' end of a full-length mouse L1. In the absence of long terminal repeats or an internal promoter, the tandem array of A-monomers may provide a mechanism for A-type L1 elements to generate transcripts containing transcriptional regulatory sequences.

Composite of A and F-type 5' terminal sequences defines a subfamily of mouse LINE-1 elements.

The 5' terminus of full-length L1 elements contains transcriptional control sequences. In mouse L1 (L1Md) elements, these sequences exist as an array of tandem direct repeats. Two types of repeat units, termed A-monomers and F-monomers, have been reported. Both monomers are about 200 bp in length but share no significant sequence homology. Previous studies have identified L1Md elements containing either A or F-monomers but not both. Here we describe three "composite" L1Md elements that contain both types of monomer sequence. Two of these composite L1Md elements are highly homologous and share the same structural rearrangements, implying that they arose from a common ancestor that has the same composite 5' end.

L1 gene conversion or same-site transposition.

DNA sequence analysis of the same chromosomal region from two haplotypes of Mus musculus and from the related species M. caroli and M. pahari reveals the presence of long interspersed sequence one (LINES-1, or L1) elements residing at the same nucleotide position in the two most distantly related of the species (M. musculus and M. pahari). The DNA sequence of each of these L1 elements is more similar to that of other L1 elements from its own species than to the other. Thus, the L1 sequence at each of these sites is recent with respect to the divergence of the species. This could be a result of recent gene conversion of L1 elements inherited from a common ancestor or of two recent independent L1 insertion events at the same nucleotide position in the two species. Such specificity of insertion would be quite different from the apparent randomness of other characterized L1 insertion events, such as those in the beta-globin locus. If the recent L1 sequences arose at this site by gene conversion of an ancestral L1 element, then the absence of an L1 element at this location in the M. caroli chromosome examined could arise either from its precise deletion from M. caroli or from the segregation into M. caroli of a polymorphic chromosome present in the ancestral population which was missing this L1 element.

Mutational analysis of human immunodeficiency virus type 1 protease suggests functional homology with aspartic proteinases.

Processing of the retroviral gag and pol gene products is mediated by a viral protease. Bacterial expression systems have been developed which permit genetic analysis of the human immunodeficiency virus type 1 protease as measured by cleavage of the pol protein precursor. Deletion analysis of the pol reading frame locates the sequences required to encode a protein with appropriate proteolytic activity near the left end of the pol reading frame but largely outside the gag-pol overlap region, which is at the extreme left end of pol. Most missense mutations within an 11-amino-acid domain highly conserved among retroviral proteases and with sequence similarity to the active site of aspartic proteinases abolish appropriate processing, suggesting that the retrovirus proteases share a catalytic mechanism with aspartic proteinases. Substitution of the amino acids flanking the scissile bond at three of the processing sites encoded by pol demonstrates distinct sequence requirements for cleavage at these different sites. The inclusion of a charged amino acid at the processing site blocks cleavage. A subset of these substitutions also inhibits processing at the nonmutated sites.