Origin of the Black Shank Resistance Gene, Ph, in Tobacco Cultivar Coker 371-Gold.

Flue-cured tobacco (Nicotiana tabacum) cultivar Coker 371-Gold (C 371-G) possesses a dominant gene, Ph, that confers high resistance to black shank disease, caused by race 0 of the soil-borne pathogen Phytophthora parasitica var. nicotianae. The origin of this gene is unknown. Breeding lines homozygous for the Ph gene were hybridized with NC 1071 and L8, flue-cured and burley genotypes known to possess qualitative resistance genes from Nicotiana plumbaginifolia and N. longiflora, respectively. The F1 hybrids were out-crossed to susceptible testers and the progenies evaluated in field black shank nurseries and in greenhouse disease tests with P. parasitica var. nicotianae race 0. Results showed that Ph was allelic to Php from N. plumbaginifolia in NC 1071. Testcross populations of hybrids between burley lines homozygous for Ph and L8, possessing Phl from N. longiflora, showed that Ph and Phl integrated into the same tobacco chromosome during interspecific transfer. Nevertheless, the two loci were estimated to be 3 cM apart. Random amplified polymorphic DNA (RAPD) analyses of the testcross progenies confirmed that recombination between the two loci was occurring. Forty-eight RAPD markers linked to Ph in doubled haploid lines were used in cluster analyses with multiple accessions of N. longiflora and N. plumbaginifolia, breeding lines L8, NC 1071, and DH92-2770-40, and cultivars K 326, Hicks, and C 371-G. A cladogram or region tree confirmed the data obtained from field and greenhouse trials, that Ph, transferred from C 371-G to DH92-2770-40, and Php in NC 1071 were allelic and originated from N. plumbaginifolia.

Phylogenetic analyses of amino acid variation in the serpin proteins.

Phylogenetic analyses of 110 serpin protein sequences revealed clades consistent with independent phylogenetic analyses based on exon-intron structure and diagnostic amino acid sites. Trees were estimated by maximum likelihood, neighbor joining, and partial split decomposition using both the BLOSUM 62 and Jones-Taylor-Thornton substitution matrices. Neighbor-joining trees gave results closest to those based on independent analyses using genomic and chromosomal data. The maximum-likelihood trees derived using the quartet puzzling algorithm were very conservative, producing many small clades that separated groups of proteins that other results suggest were related. Independent analyses based on exon-intron structure suggested that a neighbor-joining tree was more accurate than maximum-likelihood trees obtained using the quartet puzzling algorithm.

Vertebrate serpins: construction of a conflict-free phylogeny by combining exon-intron and diagnostic site analyses.

A combination of three independent biological features, genomic organization, diagnostic amino acid sites, and rare indels, was used to elucidate the phylogeny of the vertebrate serpin (serine protease inhibitor) superfamily. A strong correlation between serpin gene families displaying (1) a conserved exon-intron pattern and (2) family-specific combinations of amino acid residues at specific sites suggests that present-day vertebrates encompass six serpin gene families which evolved from primordial genes by massive intron insertion before or during early vertebrate radiation. Introns placed at homologous positions in the gene sequences in combination with diagnostic sequence characters may also constitute a reliable kinship indicator for other protein superfamilies.

Characterization and differential expression of a human gene family of olfactomedin-related proteins.

Olfactomedin-related proteins are secreted glycoproteins with conserved C-terminal motifs. Olfactomedin was originally identified as the major component of the mucus layer that surrounds the chemosensory dendrites of olfactory neurons. Homologues were subsequently found also in other tissues, including the brain and in species ranging from Caenorhabditis elegans to Homo sapiens. Most importantly, the TIGR/myocilin protein, expressed in the eye and associated with the pathogenesis of glaucoma, is an olfactomedin-related protein. The prevalence of olfactomedin-related proteins among species and their identification in different tissues prompted us to investigate whether a gene family exists within a species, specifically Homo sapiens. A GenBank search indeed revealed an entire human gene family of olfactomedin-related proteins with at least five members, designated hOlfA through hOlfD and the TIGR/myocilin protein. hOlfA corresponds to the rat neuronal AMZ protein. Phylogenetic analyses of 18 olfactomedin-related sequences resolved four distinct subfamilies. Among the human proteins, hOlfA and hOlfC, both expressed in brain, are most closely related. Northern blot analyses of 16 human tissues demonstrated highly specific expression patterns: hOlfA is expressed in brain, hOlfB in pancreas and prostate, hOlfC in cerebellum, hOlfD in colon, small intestine and prostate and TIGR/myocilin in heart and skeletal muscle. The link between TIGR/myocilin and ocular hypertension and the expression of several of these proteins in mucus-lined tissues suggest that they play an important role in regulating physical properties of the extracellular environment. Future studies can now assess whether other members of this gene family, like TIGR/myocilin, are also associated with human disease processes.

Body weight and tail length divergence in mice selected for rate of development.

A series of mouse lines has been produced by 19 generations of restricted index selection for rate of development during early and late ontogeny. The selection program was based on an index with the following four replicated selection treatments: E(+) and E(-) were selected to alter birth to 10-day body weight gain while holding late gain for both selection lines constant; correspondingly, L(+) and L(-) were selected to alter 28- to 56-day body weight gain holding early gain for both lines constant. Herein, we characterize response to selection for growth rate by analyzing age-specific mouse body weight and tail lengths and for growth curves using a logistics model. Selection on developmental rate has resulted in divergence in both age-specific and growth curve traits. E(+) and L(+) lines reached identical weights during the late selection interval, then diverged to unique mature weights. E(-) and L(-) lines similarly achieved identical weights during late selection and diverged to unique mature weights. However, the shapes of early and late growth curves were significantly divergent, and at least two distinct growth patterns are shown to result from selection. Response in body weight gain was accompanied by similar, though less pronounced, change in tail length traits. Significant response during intervals of restricted growth was also found, especially in lines selected for late gain. The evolution of the growth trajectory under restricted index selection is discussed in terms of drift and available additive genetic variation and covariation.

Cellular consequences in the brain and liver of age-specific selection for rate of development in mice.

Changes in cell number (hyperplasia) and cell size (hypertrophy) in the brain and liver are described for mice subjected to 24 generations of age-specific restricted index selection for rate of development in body weight. One selection treatment (E) altered rate of development between birth and 10 days of age, another treatment (L) involved changes in rate of development between 28 and 56 days of age, while a third control treatment (C) involved random selection. Each selection treatment was replicated three times. These age-specific selection treatments focused on intervals during ontogeny when different developmental processes (hypertrophy or hyperplasia) were more predominant in the control of growth. Significant changes in brain and liver weight occurred at both 28 and 70 days of age. Early selection (E) generated significant changes in the number of cells in the brain while later selection (L) had no effect since the brain had stopped growth before selection was initiated. For the liver, early and late selection produced significant effects on both cell number and cell size. These results describe the dynamic and multidimensional aspects of selection in terms of its ability to alter different cellular and developmental components of complex morphological traits.

Effect of long-term selection for early postnatal growth rate on survival and prenatal development of transferred mouse embryos.

Reciprocal embryo transfer procedures were performed among mouse selection lines to examine prenatal maternal effects on survival and development of transferred embryos. Mice were from generations 28 and 29 of an experiment to select for (i) increased body weight again from 0 to 10 days (E+); (ii) decreased body weight gain from 0 to 10 days (E-); or (iii) a randomly bred control line (C). A total of 118 embryo transfer procedures performed 12 h after conception resulted in 983 progeny born to 89 litters. There was a 39% overall embryo survival rate and 75% overall pregnancy success rate. Response to superovulation and oestrous synchronization was significantly lower (P < 0.01) in the E+ line. E+ individuals that did superovulate produced an average of 37 oocytes per flush, which was significantly higher than in the control line mice (29 oocytes per flush; P < 0.01). The ability to complete pregnancy successfully was not influenced by uterine environment or embryo-uterine interaction. In contrast, embryo survival in successful pregnancies was significantly affected by uterine environment. There were large maternal effects for body weight and tail length at birth; E+ recipients produced pups that were significantly larger than E- recipient pups (P < 0.01), which in turn were significantly larger than pups gestated by control recipients (P < 0.01).

Separation of phylogenetic and functional associations in biological sequences by using the parametric bootstrap.

Quantitative analyses of biological sequences generally proceed under the assumption that individual DNA or protein sequence elements vary independently. However, this assumption is not biologically realistic because sequence elements often vary in a concerted manner resulting from common ancestry and structural or functional constraints. We calculated intersite associations among aligned protein sequences by using mutual information. To discriminate associations resulting from common ancestry from those resulting from structural or functional constraints, we used a parametric bootstrap algorithm to construct replicate data sets. These data are expected to have intersite associations resulting solely from phylogeny. By comparing the distribution of our association statistic for the replicate data against that calculated for empirical data, we were able to assign a probability that two sites covaried resulting from structural or functional constraint rather than phylogeny. We tested our method by using an alignment of 237 basic helix-loop-helix (bHLH) protein domains. Comparison of our results against a solved three-dimensional structure confirmed the identification of several sites important to function and structure of the bHLH domain. This analytical procedure has broad utility as a first step in the identification of sites that are important to biological macromolecular structure and function when a solved structure is unavailable.

Correlations among amino acid sites in bHLH protein domains: an information theoretic analysis.

An information theoretic approach is used to examine the magnitude and origin of associations among amino acid sites in the basic helix-loop-helix (bHLH) family of transcription factors. Entropy and mutual information values are used to summarize the variability and covariability of amino acids comprising the bHLH domain for 242 sequences. When these quantitative measures are integrated with crystal structure data and summarized using helical wheels, they provide important insights into the evolution of three-dimensional structure in these proteins. We show that amino acid sites in the bHLH domain known to pack against each other have very low entropy values, indicating little residue diversity at these contact sites. Noncontact sites, on the other hand, exhibit significantly larger entropy values, as well as statistically significant levels of mutual information or association among sites. High levels of mutual information indicate significant amounts of intercorrelation among amino acid residues at these various sites. Using computer simulations based on a parametric bootstrap procedure, we are able to partition the observed covariation among various amino acid sites into that arising from phylogenetic (common ancestry) and stochastic causes and those resulting from structural and functional constraints. These results show that a significant amount of the observed covariation among amino acid sites is due to structural/functional constraints, over and above the covariation arising from phylogenetic constraints. These quantitative analyses provide a highly integrated evolutionary picture of the multidimensional dynamics of sequence diversity and protein structure.

Molecular evolution of the GATA family of transcription factors: conservation within the DNA-binding domain.

The GATA-binding transcription factors comprise a protein family whose members contain either one or two highly conserved zinc finger DNA-binding domains. Members of this group have been identified in organisms ranging from cellular slime mold to vertebrates, including plants, fungi, nematodes, insects, and echinoderms. While much work has been done describing the expression patterns, functional aspects, and target genes for many of these proteins, an evolutionary analysis of the entire family has been lacking. Herein we show that only the C-terminal zinc finger (Cf) and basic domain, which together constitute the GATA-binding domain, are conserved throughout this protein family. Phylogenetic analyses of amino acid sequences demonstrate distinct evolutionary pathways. Analysis of GATA factors isolated from vertebrates suggests that the six distinct vertebrate GATAs are descended from a common ancestral sequence, while those isolated from nonvertebrates (with the exception of the fungal AREA orthologues and Arabidopsis paralogues) appear to be related only within the DNA-binding domain and otherwise provide little insight into their evolutionary history. These results suggest multiple modes of evolution, including gene duplication and modular evolution of GATA factors based upon inclusion of a class IV zinc finger motif. As such, GATA transcription factors represent a group of proteins related solely by their homologous DNA-binding domains. Further analysis of this domain examines the degree of conservation at each amino acid site using the Boltzmann entropy measure, thereby identifying residues critical to preservation of structure and function. Finally, we construct a predictive motif that can accurately identify potential GATA proteins.

Evolution of bHLH transcription factors: modular evolution by domain shuffling?

Multidomain proteins usually contain several conserved and apparently independently evolved domains. As a result, classifications based on only a single small domain may obscure the true evolutionary relationships of the proteins. The current classification of basic helix-loop-helix (bHLH) domain-containing proteins is based on the conserved bHLH domain alone. Herein, we explore whether sequence homology and, therefore, evolutionary relationships can be detected among the flanking or non-bHLH components of the amino acid sequences of 122 bHLH proteins. These 122 proteins were the same proteins previously used to construct the existing classification of the bHLH-domain-containing proteins. Several possible scenarios are examined in order to explain the observed patterns of sequence divergence, including (1) monophyly, (2) convergent evolution, (3) addition of functional components to the bHLH domain, and (4) modular evolution with domain shuffling. Drawing on several lines of evidence, we suggest that modular evolution by domain shuffling may have played an important role in the evolution of this large group of transcriptional regulators.

Uterine and postnatal maternal effects in mice selected for differential rate of early development.

A series of mouse lines was produced by long-term restricted index selection for divergent rate of growth during early and late postnatal development. The selection program was based on the following treatments: E(+) and E(-) lines were selected to alter birth to 10-day weight gain while holding late gain for both lines constant and a control line was established via random selection. Using embryo transfer and crossfostering methodology, we partitioned postnatal growth for E(+), E(-), and C lines into progeny genetic, uterine maternal, and nurse maternal components. Selection for differential early growth resulted in correlated response in uterine and nurse maternal effects on body weights, with significant genetic-by-environment interactions. Significant uterine effects were also observed in tail length measurements. Direct uterine effects on body weight were relatively small and resulted in growth rate differences early in development. Nurse effects were large, resulting in modification of progeny growth trajectory especially during early postnatal development. Genetic-by-uterine interactions were large and demonstrate progeny-specific effects of the prenatal uterine environment.

Effect of selection for development rate on reproductive onset in female mice.

This research reports analyses of correlated response in reproductive onset in ICR mice after 23 generations of restricted index selection for divergent body weight gain, early (birth-10 days) or later (28-56 days) in life. Long-term selection altered growth trajectories and 56 day body weight of individuals under different selection regimes in this study. Mice in lines under early selection have the same percentage mature weight at vaginal opening as controls (63%). Vaginal opening is delayed in mice selected for slow early growth, which take longer to reach what appears to be a weight threshold. In contrast, individuals in lines selected for later slow growth undergo vaginal opening at the same age as controls, but at a lower weight and increased percentage mature weight. Pre-compensation or 'counter-balance growth' is observed in these lines, with mice selected for late enhanced growth reaching 52% of mature weight at vaginal opening while mice with late slow growth attain 71% of mature weight prior to vaginal opening. Only 42% of mice with late slow growth attain first oestrus by 56 days. We speculate this is a function of growth rate and fat/lean ratio. Mice with early slow growth show compensatory growth, reaching first oestrus at a similar time to controls. We conclude that selection for growth rate has asymmetrically affected reproductive onset, with lines selected for suppressed gains experiencing delays in the reproductive onset traits measured.

Molecular evolution of helix-turn-helix proteins.

The helix-turn-helix domain-containing family of transcriptional regulators is of ancient origin and has been incorporated into numerous disparate biological processes. As a consequence, the forces shaping its early evolution have been difficult to reconstruct. Herein, we analyze this large and diverse family with a combination of traditional phylogenetic techniques and newer sequence analysis tools to determine whether the helix-turn-helix family arose from a single common ancestor. Our analyses of the DNA-binding domain show that amino acid chemistry is conserved at many sites in the first helix and the turn. The high level of divergence combined with the short length of the domain hinders robust reconstruction of the entire phylogeny, but some level of deep node inference is possible. All analyses point to a predominantly monophyletic origin for the helix-turn-helix domain. The consequences of such an origin for a diverse group of proteins, and guidelines for the identification of future members of the HTH family are discussed.

Positional dependence, cliques, and predictive motifs in the bHLH protein domain.

Quantitative analyses were carried out on a large number of proteins that contain the highly conserved basic helix-loop-helix domain. Measures derived from information theory were used to examine the extent of conservation at amino acid sites within the bHLH domain as well as the extent of mutual information among sites within the domain. Using the Boltzmann entropy measure, we described the extent of amino acid conservation throughout the bHLH domain. We used position association (pa) statistics that reflect the joint probability of occurrence of events to estimate the "mutual information content" among distinct amino acid sites. Further, we used pa statistics to estimate the extent of association in amino acid composition at each site in the domain and between amino acid composition and variables reflecting clade and group membership, loop length, and the presence of a leucine zipper. The pa values were also used to describe groups of amino acid sites called "cliques" that were highly associated with each other. Finally, a predictive motif was constructed that accurately identifies bHLH domain-containing proteins that belong to Groups A and B.

Segment-based scores for pairwise and multiple sequence alignments.

In this paper, we discuss a novel scoring scheme for sequence alignments. The score of an alignment is defined as the sum of so-called weights of aligned segment pairs. A simple modification of the weight function used by the original version of the DIALIGN alignment program turns out to have a crucial advantage: it can be applied to both, global and local alignment problems without the need to specify a threshold parameter.

Molecular evolution of the Myb family of transcription factors: evidence for polyphyletic origin.

The Myb family of proteins is a group of functionally diverse transcriptional activators found in both plants and animals that is characterized by a conserved DNA-binding domain of approximately 50 amino acids. Phylogenetic analyses of amino acid sequences of this family of proteins portray very disparate evolutionary histories in plants and animals. Animal Myb proteins have diverged from a common ancestor, while plants appear related only within the DNA-binding domain. Results imply a pattern of modular evolution of the Myb proteins centering on the possession of a helix-turn-helix motif. Based on this it is suggested that Myb proteins are a polyphyletic group related only by a "Myb-box" DNA-binding motif.

Developmental quantitative genetics, conditional epigenetic variability and growth in mice.

Ontogenetic variation in the causal components of phenotypic variability and covariability is described for body weight and tail length in mice derived from a full 7 x 7 diallel cross. Age-related changes in additive, dominance, sex-linked and maternal variance and covariance between 14 and 70 days of age are described. Age-specific variance components at time t are conditioned on the causal genetic effects at time (t - 1). This procedure demonstrates the generation of significant episodes of new genetic variation arising at specific intervals during ontogeny. These episodes of new genetic variation are placed in the context of epigenetic models in developmental quantitative genetics. These results are also concordant on recent findings on age-specific gene expression in mouse growth as shown by QTL analyses.

Altering developmental trajectories in mice by restricted index selection.

A restricted index selection experiment on mice was carried out for 1-4 generations on rate of early postnatal development (growth rate from birth to 10 days of age) vs. rate of development much later in ontogeny (growth rate from 28 to 56 days of age). Early rate of development (E) approximates hyperplasia (changes in cell number) and later rate (L) reflects hypertropy (changes in cell size). The selection criteria were as follows; E+LO was selected to increase early body weight gain while holding late body weight gain constant; E-LO was selected to decrease early body gain while holding late gain constant; EOL+ was selected to increase late gain holding early gain constant; and EOL- was selected to decrease late gain holding early gain constant. After 14 generations of selection, significant divergence among lines has occurred and the changes in the growth trajectories are very close to expectation. The genetic and developmental bases of complex traits are discussed as well as the concept of developmental homoplasy.

A natural classification of the basic helix-loop-helix class of transcription factors.

A natural (evolutionary) classification is provided for 242 basic helix-loop-helix (bHLH) motif-containing proteins. Phylogenetic analyses of amino acid sequences describe the patterns of evolutionary change within the motif and delimit evolutionary lineages. These evolutionary lineages represent well known functional groups of proteins and can be further arranged into five groups based on binding to DNA at the hexanucleotide E-box, the amino acid patterns in other components of the motif, and the presence/absence of a leucine zipper. The hypothesized ancestral amino acid sequence for the bHLH transcription factor family is given together with the ancestral sequences of the subgroups. It is suggested that bHLH proteins containing a leucine zipper are not a natural, monophyletic group.