Phylogenetic analysis of “Volvocacae” for comparative genetic studies

Sequence analysis based on multiple isolates representing essentially all genera and species of the classic family Volvocaeae has clarified their phylogenetic relationships. Cloned internal transcribed spacer sequences (ITS-1 and ITS-2, flanking the 5.8S gene of the nuclear ribosomal gene cistrons) were aligned, guided by ITS transcript secondary structural features, and subjected to parsimony and neighbor joining distance analysis. Results confirm the notion of a single common ancestor, and Chlamydomonas reinharditii alone among all sequenced green unicells is most similar. Interbreeding isolates were nearest neighbors on the evolutionary tree in all cases. Some taxa, at whatever level, prove to be clades by sequence comparisons, but others provide striking exceptions. The morphological species Pandorina morum, known to be widespread and diverse in mating pairs, was found to encompass all of the isolates of the four species of Volvulina. Platydorina appears to have originated early and not to fall within the genus Eudorina, with which it can sometimes be confused by morphology. The four species of Pleodorina appear variously associated with Eudorina examples. Although the species of Volvox are each clades, the genus Volvox is not. The conclusions confirm and extend prior, more limited, studies on nuclear SSU and LSU rDNA genes and plastid-encoded rbcL and atpB. The phylogenetic tree suggests which classical taxonomic characters are most misleading and provides a framework for molecular studies of the cell cycle-related and other alterations that have engendered diversity in both vegetative and sexual colony patterns in this classical family.

Electrostatic interaction between helical macromolecules in dense aggregates: An impetus for DNA poly- and meso-morphism

DNA exhibits a surprising multiplicity of structures when it is packed into dense aggregates. It undergoes various polymorphous transitions (e.g., from the B to A form) and mesomorphous transformations (from hexagonal to orthorhombic or monoclinic packing, changes in the mutual alignment of nearest neighbors, etc). In this report we show that such phenomena may have their origin in the specific helical symmetry of the charge distribution on DNA surface. Electrostatic interaction between neighboring DNA molecules exhibits strong dependence on the patterns of molecular surface groups and adsorbed counter-ions. As a result, it is affected by such structural parameters as the helical pitch, groove width, the number of base pairs per helical turn, etc. We derive expressions which relate the energy of electrostatic interaction with these parameters and with the packing variables characterizing the axial and azimuthal alignment between neighboring macromolecules. We show, in particular, that the structural changes upon the B-to-A transition reduce the electrostatic energy by ≈kcal/mol per base pair, at a random adsorption of counter ions. Ion binding into the narrow groove weakens or inverts this effect, stabilizing B-DNA, as it is presumably the case in Li+-DNA assemblies. The packing symmetry and molecular alignment in DNA aggregates are shown to be affected by the patterns of ion binding.

Non-CpG methylation is prevalent in embryonic stem cells and may be mediated by DNA methyltransferase 3a

Current evidence indicates that methylation of cytosine in mammalian DNA is restricted to both strands of the symmetrical sequence CpG, although there have been sporadic reports that sequences other than CpG may also be methylated. We have used a dual-labeling nearest neighbor technique and bisulphite genomic sequencing methods to investigate the nearest neighbors of 5-methylcytosine residues in mammalian DNA. We find that embryonic stem cells, but not somatic tissues, have significant cytosine-5 methylation at CpA and, to a lesser extent, at CpT. As the expression of the de novo methyltransferase Dnmt3a correlates well with the presence of non-CpG methylation, we asked whether Dnmt3a might be responsible for this modification. Analysis of genomic methylation in transgenic Drosophila expressing Dnmt3a reveals that Dnmt3a is predominantly a CpG methylase but also is able to induce methylation at CpA and at CpT.

How are close residues of protein structures distributed in primary sequence?

Structurally neighboring residues are categorized according to their separation in the primary sequence as proximal (1-4 positions apart) and otherwise distal, which in turn is divided into near (5-20 positions), far (21-50 positions), very far ( > 50 positions), and interchain (from different chains of the same structure). These categories describe the linear distance histogram (LDH) for three-dimensional neighboring residue types. Among the main results are the following: (i) nearest-neighbor hydrophobic residues tend to be increasingly distally separated in the linear sequence, thus most often connecting distinct secondary structure units. (ii) The LDHs of oppositely charged nearest-neighbors emphasize proximal positions with a subsidiary maximum for very far positions. (iii) Cysteine-cysteine structural interactions rarely involve proximal positions. (iv) The greatest numbers of interchain specific nearest-neighbors in protein structures are composed of oppositely charged residues. (v) The largest fraction of side-chain neighboring residues from beta-strands involves near positions, emphasizing associations between consecutive strands. (vi) Exposed residue pairs are predominantly located in proximal linear positions, while buried residue pairs principally correspond to far or very far distal positions. The results are principally invariant to protein sizes, amino acid usages, linear distance normalizations, and over- and underrepresentations among nearest-neighbor types. Interpretations and hypotheses concerning the LDHs, particularly those of hydrophobic and charged pairings, are discussed with respect to protein stability and functionality. The pronounced occurrence of oppositely charged interchain contacts is consistent with many observations on protein complexes where multichain stabilization is facilitated by electrostatic interactions.

Cultural variation in Africa: role of mechanisms of transmission and adaptation.

Cultural inheritance can be considered as a mechanism of adaptation made possible by communication, which has reached its greatest development in humans and can allow long-term conservation or rapid change of culturally transmissible traits depending on circumstances and needs. Conservativeness/flexibility is largely modulated by mechanisms of sociocultural transmission. An analysis was carried out by testing the fit of three models to 47 cultural traits (classified in six groups) in 277 African societies. Model A (demic diffusion) is conservation over generations, as shown by correlations of cultural traits with language, used as a measure of historical connection. Model B (environmental adaptation) is measured by correlation to the natural environment. Model C (cultural diffusion) is the spread to neighbors by social contact in an epidemic-like fashion and was tested by measuring the tightness of geographic clustering of the traits. Most traits examined, in particular those affecting family structure and kinship, showed great conservation over generations, as shown by the fit of model A. They are most probably transmitted by family members. This is in agreement with the theoretical demonstration that cultural transmission in the family (vertical) is the most conservative one. Some traits show environmental effects, indicating the importance of adaptation to physical environment. Only a few of the 47 traits showed tight geographic clustering indicating that their spread to nearest neighbors follows model C, as is usually the case for transmission among unrelated people (called horizontal transmission).

A new method for generating point mutations in bacterial artificial chromosomes by homologous recombination in Escherichia coli

Bacterial artificial chromosomes (BACs) and P1 artificial chromosomes (PACs), which contain large fragments of genomic DNA, have been successfully used as transgenes to create mouse models of dose-dependent diseases. They are also potentially valuable as transgenes for dominant diseases given that point mutations and/or small rearrangements can be accurately introduced. Here, we describe a new method to introduce small alterations in BACs, which results in the generation of point mutations with high frequency. The method involves homologous recombination between the original BAC and a shuttle vector providing the mutation. Each recombination step is monitored using positive and negative selection markers, which are the Kanamycin-resistance gene, the sacB gene and temperature-sensitive replication, all conferred by the shuttle plasmid. We have used this method to introduce four different point mutations and the insertion of the β-galactosidase gene in a BAC, which has subsequently been used for transgenic animal production.

Random circular permutation of genes and expressed polypeptide chains: application of the method to the catalytic chains of aspartate transcarbamoylase.

Recent studies on proteins whose N and C termini are in close proximity have demonstrated that folding of polypeptide chains and assembly of oligomers can be accomplished with circularly permuted chains. As yet no methodical study has been conducted to determine how extensively new termini can be introduced and where such termini cannot be tolerated. We have devised a procedure to generate random circular permutations of the catalytic chains of Escherichia coli aspartate transcarbamoylase (ATCase; EC 2.1.3.2) and to select clones that produce active or stable holoenzyme containing permuted chains. A tandem gene construct was made, based on the desired linkage between amino acid residues in the C- and N-terminal regions of the polypeptide chain, and this DNA was treated with a suitable restriction enzyme to yield a fragment containing the rearranged coding sequence for the chain. Circularization achieved with DNA ligase, followed by linearization at random with DNase I, and incorporation of the linearized, repaired, blunt-ended, rearranged genes into a suitable plasmid permitted the expression of randomly permuted polypeptide chains. The plasmid with appropriate stop codons also contained pyrI, the gene encoding the regulatory chain of ATCase. Colonies expressing detectable amounts of ATCase-like molecules containing permuted catalytic chains were identified by an immunoblot technique or by their ability to grow in the absence of pyrimidines in the growth medium. Sequencing of positive clones revealed a variety of novel circular permutations. Some had N and C termini within helices of the wild-type enzyme as well as deletions and insertions. Permutations were concentrated in the C-terminal domain and only few were detected in the N-terminal domain. The technique, which is adaptable generally to proteins whose N and C termini are near each other, can be of value in relating in vivo folding of nascent, growing polypeptide chains to in vitro renaturation of complete chains and determining the role of protein sequence in folding kinetics.

An iterative method for extracting energy-like quantities from protein structures.

We present a method (ENERGI) for extracting energy-like quantities from a data base of protein structures. In this paper, we use the method to generate pairwise additive amino acid "energy" scores. These scores are obtained by iteration until they correctly discriminate a set of known protein folds from decoy conformations. The method succeeds in lattice model tests and in the gapless threading problem as defined by Maiorov and Crippen [Maiorov, V. N. & Crippen, G. M. (1992) J. Mol. Biol. 227, 876-888]. A more challenging test of threading a larger set of test proteins derived from the representative set of Hobohm and Sander [Hobohm, U. & Sander, C. (1994) Protein Sci. 3, 522-524] is used as a "workbench" for exploring how the ENERGI scores depend on their parameter sets.

Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries.

A new and highly effective method, termed suppression subtractive hybridization (SSH), has been developed for the generation of subtracted cDNA libraries. It is based primarily on a recently described technique called suppression PCR and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of cDNAs within the target population and the subtraction step excludes the common sequences between the target and driver populations. In a model system, the SSH technique enriched for rare sequences over 1,000-fold in one round of subtractive hybridization. We demonstrate its usefulness by generating a testis-specific cDNA library and by using the subtracted cDNA mixture as a hybridization probe to identify homologous sequences in a human Y chromosome cosmid library. The human DNA inserts in the isolated cosmids were further confirmed to be expressed in a testis-specific manner. These results suggest that the SSH technique is applicable to many molecular genetic and positional cloning studies for the identification of disease, developmental, tissue-specific, or other differentially expressed genes.

Direct detection and isolation of restriction landmark genomic scanning (RLGS) spot DNA markers tightly linked to a specific trait by using the RLGS spot-bombing method.

We have developed a technique for isolating DNA markers tightly linked to a target region that is based on RLGS, named RLGS spot-bombing (RLGS-SB). RLGS-SB allows us to scan the genome of higher organisms quickly and efficiently to identify loci that are linked to either a target region or gene of interest. The method was initially tested by analyzing a C57BL/6-GusS mouse congenic strain. We identified 33 variant markers out of 10,565 total loci in a 4.2-centimorgan (cM) interval surrounding the Gus locus in 4 days of laboratory work. The validity of RLGS-SB to find DNA markers linked to a target locus was also tested on pooled DNA from segregating backcross progeny by analyzing the spot intensity of already mapped RLGS loci. Finally, we used RLGS-SB to identify DNA markers closely linked to the mouse reeler (rl) locus on chromosome 5 by phenotypic pooling. A total of 31 RLGS loci were identified and mapped to the target region after screening 8856 loci. These 31 loci were mapped within 11.7 cM surrounding rl. The average density of RLGS loci located in the rl region was 0.38 cM. Three loci were closely linked to rl showing a recombination frequency of 0/340, which is < 1 cM from rl. Thus, RLGS-SB provides an efficient and rapid method for the detection and isolation of polymorphic DNA markers linked to a trait or gene of interest.