Naturally occurring variation in copia expression is due to both element (cis) and host (trans) regulatory variation.

Significant differences in levels of copia [Drosophila long terminal repeat (LTR) retrotransposon] expression exist among six species representing the Drosophila melanogaster species complex (D. melanogaster, Drosophila mauritiana, Drosophila simulans, Drosophila sechellia, Drosophila yakuba, and Drosophila erecta) and a more distantly related species (Drosophila willistoni). These differences in expression are correlated with major size variation mapping to putative regulatory regions of the copia 5' LTR and adjacent untranslated leader region (ULR). Sequence analysis indicates that these size variants were derived from a series of regional duplication events. The ability of the copia LTR-ULR size variants to drive expression of a bacterial chloramphenicol acetyltransferase reporter gene was tested in each of the seven species. The results indicate that both element-encoded (cis) and host-genome-encoded (trans) genetic differences are responsible for the variability in copia expression within and between Drosophila species. This finding indicates that models purporting to explain the dynamics and distribution of retrotransposons in natural populations must consider the potential impact of both element-encoded and host-genome-encoded regulatory variation to be valid. We propose that interelement selection among retrotransposons may provide a molecular drive mechanism for the evolution of eukaryotic enhancers which can be subsequently distributed throughout the genome by retrotransposition.

Incipient species formation in salamanders of the Ensatina complex

The Ensatina eschscholtzii complex of plethodontid salamanders, a well-known “ring species,” is thought to illustrate stages in the speciation process. Early research, based on morphology and coloration, has been extended by the incorporation of studies of protein variation and mitochondrial DNA sequences. The new data show that the complex includes a number of geographically and genetically distinct components that are at or near the species level. The complex is old and apparently has undergone instances of range contraction, isolation, differentiation, and then expansion and secondary contact. While the hypothesis that speciation is retarded by gene flow around the ring is not supported by molecular data, the general biogeographical hypothesis is supported. There is evidence of a north to south range expansion along two axes, with secondary contact and completion of the ring in southern California. Current research targets regions once thought to show primary intergradation, but which molecular markers reveal to be zones of secondary contact. Here emphasis is on the subspecies E. e. xanthoptica, which is involved in four distinct secondary contacts in central California. There is evidence of renewed genetic interactions upon recontact, with greater genetic differentiation within xanthoptica than between it and some of the interacting populations. The complex presents a full array of intermediate conditions between well-marked species and geographically variable populations. Geographically differentiated segments represent a diversity of depths of time of isolation and admixture, reflecting the complicated geomorphological history of California. Ensatina illustrates the continuing difficulty in making taxonomic assignments in complexes studied during species formation.

Diversification, expression, and γδ T cell recognition of evolutionarily distant members of the MIC family of major histocompatibility complex class I-related molecules

Distant relatives of major histocompatibility complex (MHC) class I molecules, human MICA and MICB, function as stress-induced antigens that are broadly recognized by intestinal epithelial γδ T cells. They may thus play a central role in the immune surveillance of damaged, infected, or otherwise stressed intestinal epithelial cells. However, the generality of this system in evolution and the mode of recognition of MICA and MICB are undefined. Analysis of cDNA sequences from various primate species defined translation products that are homologous to MICA and MICB. All of the MIC polypeptides have common characteristics, although they are extraordinarily diverse. The most notable alterations are several deletions and frequent amino acid substitutions in the putative α-helical regions of the α1α2 domains. However, the primate MIC molecules were expressed on the surfaces of normal and transfected cells. Moreover, despite their sharing of relatively few identical amino acids in potentially accessible regions of their α1α2 domains, they were recognized by diverse human intestinal epithelial γδ T cells that are restricted by MICA and MICB. Thus, MIC molecules represent a family of MHC proteins that are structurally diverse yet appear to be functionally conserved. The promiscuous mode of γδ T cell recognition of these antigens may be explained by their sharing of a single conserved interaction site.

Crystal structure of Taq DNA polymerase in complex with an inhibitory Fab: The Fab is directed against an intermediate in the helix-coil dynamics of the enzyme

We report the crystal structure of Thermus aquaticus DNA polymerase I in complex with an inhibitory Fab, TP7, directed against the native enzyme. Some of the residues present in a helical conformation in the native enzyme have adopted a γ turn conformation in the complex. Taken together, structural information that describes alteration of helical structure and solution studies that demonstrate the ability of TP7 to inhibit 100% of the polymerase activity of the enzyme suggest that the change in conformation is probably caused by trapping of an intermediate in the helix-coil dynamics of this helix by the Fab. Antibodies directed against modified helices in proteins have long been anticipated. The present structure provides direct crystallographic evidence. The Fab binds within the DNA binding cleft of the polymerase domain, interacting with several residues that are used by the enzyme in binding the primer:template complex. This result unequivocally corroborates inferences drawn from binding experiments and modeling calculations that the inhibitory activity of this Fab is directly attributable to its interference with DNA binding by the polymerase domain of the enzyme. The combination of interactions made by the Fab residues in both the polymerase and the vestigial editing nuclease domain of the enzyme reveal the structural basis of its preference for binding to DNA polymerases of the Thermus species. The orientation of the structure-specific nuclease domain with respect to the polymerase domain is significantly different from that seen in other structures of this polymerase. This reorientation does not appear to be antibody-induced and implies remarkably high relative mobility between these two domains.

The newt ribozyme is part of a riboprotein complex

Strand-specific transcripts of a satellite DNA of the newts, Notophthalmus and Triturus, are present in cells in monomeric and multimeric sizes. These transcripts undergo self-catalyzed, site-specific cleavage in vitro: the reaction requires Mg2+ and is mediated by a “hammerhead” domain. Transcription of the newt ribozyme appears to be performed by RNA polymerase II under the control of a proximal sequence element and a distal sequence element. In vitro, the newt ribozyme can cleave in trans an RNA substrate, suggesting that in vivo it might be involved in RNA processing events, perhaps as a riboprotein complex. Here we show that the newt ribozyme is in fact present as a riboprotein particle of about 12 S in the oocytes of Triturus. In addition, reconstitution experiments and gel-shift analyses show that a complex is assembled in vitro on the monomeric ribozyme molecules. UV cross-linking studies identify a few polypeptide species, ranging from 31 to 65 kDa, associated to the newt ribozyme with different affinities. Finally, we find that an appropriate oligoribonucleotide substrate is specifically cleaved by the riboproteic activity in S-100 ovary extracts.

Restricted structural gene polymorphism in the Mycobacterium tuberculosis complex indicates evolutionarily recent global dissemination

One-third of humans are infected with Mycobacterium tuberculosis, the causative agent of tuberculosis. Sequence analysis of two megabases in 26 structural genes or loci in strains recovered globally discovered a striking reduction of silent nucleotide substitutions compared with other human bacterial pathogens. The lack of neutral mutations in structural genes indicates that M. tuberculosis is evolutionarily young and has recently spread globally. Species diversity is largely caused by rapidly evolving insertion sequences, which means that mobile element movement is a fundamental process generating genomic variation in this pathogen. Three genetic groups of M. tuberculosis were identified based on two polymorphisms that occur at high frequency in the genes encoding catalase-peroxidase and the A subunit of gyrase. Group 1 organisms are evolutionarily old and allied with M. bovis, the cause of bovine tuberculosis. A subset of several distinct insertion sequence IS6110 subtypes of this genetic group have IS6110 integrated at the identical chromosomal insertion site, located between dnaA and dnaN in the region containing the origin of replication. Remarkably, study of ≈6,000 isolates from patients in Houston and the New York City area discovered that 47 of 48 relatively large case clusters were caused by genotypic group 1 and 2 but not group 3 organisms. The observation that the newly emergent group 3 organisms are associated with sporadic rather than clustered cases suggests that the pathogen is evolving toward a state of reduced transmissability or virulence.

Evolution of plastid gene rps2 in a lineage of hemiparasitic and holoparasitic plants: Many losses of photosynthesis and complex patterns of rate variation

The plastid genomes of some nonphotosynthetic parasitic plants have experienced an extreme reduction in gene content and an increase in evolutionary rate of remaining genes. Nothing is known of the dynamics of these events or whether either is a direct outcome of the loss of photosynthesis. The parasitic Scrophulariaceae and Orobanchaceae, representing a continuum of heterotrophic ability ranging from photosynthetic hemiparasites to nonphotosynthetic holoparasites, are used to investigate these issues. We present a phylogenetic hypothesis for parasitic Scrophulariaceae and Orobanchaceae based on sequences of the plastid gene rps2, encoding the S2 subunit of the plastid ribosome. Parasitic Scrophulariaceae and Orobanchaceae form a monophyletic group in which parasitism can be inferred to have evolved once. Holoparasitism has evolved independently at least five times, with certain holoparasitic lineages representing single species, genera, and collections of nonphotosynthetic genera. Evolutionary loss of the photosynthetic gene rbcL is limited to a subset of holoparasitic lineages, with several holoparasites retaining a full length rbcL sequence. In contrast, the translational gene rps2 is retained in all plants investigated but has experienced rate accelerations in several hemi- as well as holoparasitic lineages, suggesting that there may be substantial molecular evolutionary changes to the plastid genome of parasites before the loss of photosynthesis. Independent patterns of synonymous and nonsynonymous rate acceleration in rps2 point to distinct mechanisms underlying rate variation in different lineages. Parasitic Scrophulariaceae (including the traditional Orobanchaceae) provide a rich platform for the investigation of molecular evolutionary process, gene function, and the evolution of parasitism.

In silico detection of control signals: mRNA 3′-end-processing sequences in diverse species

We have investigated mRNA 3′-end-processing signals in each of six eukaryotic species (yeast, rice, arabidopsis, fruitfly, mouse, and human) through the analysis of more than 20,000 3′-expressed sequence tags. The use and conservation of the canonical AAUAAA element vary widely among the six species and are especially weak in plants and yeast. Even in the animal species, the AAUAAA signal does not appear to be as universal as indicated by previous studies. The abundance of single-base variants of AAUAAA correlates with their measured processing efficiencies. As found previously, the plant polyadenylation signals are more similar to those of yeast than to those of animals, with both common content and arrangement of the signal elements. In all species examined, the complete polyadenylation signal appears to consist of an aggregate of multiple elements. In light of these and previous results, we present a broadened concept of 3′-end-processing signals in which no single exact sequence element is universally required for processing. Rather, the total efficiency is a function of all elements and, importantly, an inefficient word in one element can be compensated for by strong words in other elements. These complex patterns indicate that effective tools to identify 3′-end-processing signals will require more than consensus sequence identification.

Proton uptake by bacterial reaction centers: The protein complex responds in a similar manner to the reduction of either quinone acceptor

In bacterial photosynthetic reaction centers, the protonation events associated with the different reduction states of the two quinone molecules constitute intrinsic probes of both the electrostatic interactions and the different kinetic events occurring within the protein in response to the light-generated introduction of a charge. The kinetics and stoichiometries of proton uptake on formation of the primary semiquinone QA− and the secondary acceptor QB− after the first and second flashes have been measured, at pH 7.5, in reaction centers from genetically modified strains and from the wild type. The modified strains are mutated at the L212Glu and/or at the L213Asp sites near QB; some of them carry additional mutations distant from the quinone sites (M231Arg → Leu, M43Asn → Asp, M5Asn → Asp) that compensate for the loss of L213Asp. Our data show that the mutations perturb the response of the protein system to the formation of a semiquinone, how distant compensatory mutations can restore the normal response, and the activity of a tyrosine residue (M247Ala → Tyr) in increasing and accelerating proton uptake. The data demonstrate a direct correlation between the kinetic events of proton uptake that are observed with the formation of either QA− or QB−, suggesting that the same residues respond to the generation of either semiquinone species. Therefore, the efficiency of transferring the first proton to QB is evident from examination of the pattern of H+/QA− proton uptake. This delocalized response of the protein complex to the introduction of a charge is coordinated by an interactive network that links the Q− species, polarizable residues, and numerous water molecules that are located in this region of the reaction center structure. This could be a general property of transmembrane redox proteins that couple electron transfer to proton uptake/release reactions.

Bicarbonate is an essential constituent of the water-oxidizing complex of photosystem II

It is shown that restoration of photoinduced electron flow and O2 evolution with Mn2+ in Mn-depleted photosystem II (PSII) membrane fragments isolated from spinach chloroplasts is considerably increased with bicarbonate in the region pH 5.0–8.0 in bicarbonate-depleted medium. In buffered solutions equilibrated with the atmosphere (nondepleted of bicarbonate), the bicarbonate effect is observed only at pH lower than the pK of H2CO3 dissociation (6.4), which indicates that HCO3− is the essential species for the restoration effect. The addition of just 2 Mn2+ atoms per one PSII reaction center is enough for the maximal reactivation when bicarbonate is present in the medium. Analysis of bicarbonate concentration dependence of the restoration effect reveals two binding sites for bicarbonate with apparent dissociation constant (Kd) of ≈2.5 μM and 20–34 μM when 2,6-dichloro-p-benzoquinone is used as electron acceptor, while in the presence of silicomolybdate only the latter one remains. Similar bicarbonate concentration dependence of O2 evolution was obtained in untreated Mn-containing PSII membrane fragments. It is suggested that the Kd of 20–34 μM is associated with the donor side of PSII while the location of the lower Kd binding site is not quite clear. The conclusion is made that bicarbonate is an essential constituent of the water-oxidizing complex of PSII, important for its assembly and maintenance in the functionally active state.

Murine Nkg2d and Cd94 are clustered within the natural killer complex and are expressed independently in natural killer cells

Natural killer (NK) cells express C-type lectin-like receptors, encoded in the NK gene complex, that interact with major histocompatibility complex class I and either inhibit or activate functional activity. Human NK cells express heterodimers consisting of CD94 and NKG2 family molecules, whereas murine NK cells express homodimers belonging to the Ly-49 family. The corresponding orthologues for other species, however, have not been described. In this report, we used probes derived from the expressed sequence tag database to clone C57BL/6-derived cDNAs homologous to human NKG2-D and CD94. Among normal tissues, murine NKG2-D and CD94 transcripts are highly expressed only in activated NK cells, including both Ly-49A+ and Ly-49A− subpopulations. Additionally, mNKG2-D is expressed in murine NK cell clones KY-1 and KY-2, whereas mCD94 expression is observed only in KY-1 cells but not KY-2. Last, we have finely mapped the physical location of the Cd94 (centromeric) and Nkg2d (telomeric) genes between Cd69 and the Ly49 cluster in the NK complex. Thus, these data indicate the expanding complexity of the NK complex and the corresponding repertoire of C-type lectin-like receptors on murine NK cells.

IMGT/HLA Database—a sequence database for the human major histocompatibility complex

The IMGT/HLA Database (www.ebi.ac.uk/imgt/hla/) specialises in sequences of polymorphic genes of the HLA system, the human major histocompatibility complex (MHC). The HLA complex is located within the 6p21.3 region on the short arm of human chromosome 6 and contains more than 220 genes of diverse function. Many of the genes encode proteins of the immune system and these include the 21 highly polymorphic HLA genes, which influence the outcome of clinical transplantation and confer susceptibility to a wide range of non-infectious diseases. The database contains sequences for all HLA alleles officially recognised by the WHO Nomenclature Committee for Factors of the HLA System and provides users with online tools and facilities for their retrieval and analysis. These include allele reports, alignment tools and detailed descriptions of the source cells. The online IMGT/HLA submission tool allows both new and confirmatory sequences to be submitted directly to the WHO Nomenclature Committee. The latest version (release 1.7.0 July 2000) contains 1220 HLA alleles derived from over 2700 component sequences from the EMBL/GenBank/DDBJ databases. The HLA database provides a model which will be extended to provide specialist databases for polymorphic MHC genes of other species.

Microbial phyllosphere populations are more complex than previously realized

Phyllosphere microbial communities were evaluated on leaves of field-grown plant species by culture-dependent and -independent methods. Denaturing gradient gel electrophoresis (DGGE) with 16S rDNA primers generally indicated that microbial community structures were similar on different individuals of the same plant species, but unique on different plant species. Phyllosphere bacteria were identified from Citrus sinesis (cv. Valencia) by using DGGE analysis followed by cloning and sequencing of the dominant rDNA bands. Of the 17 unique sequences obtained, database queries showed only four strains that had been described previously as phyllosphere bacteria. Five of the 17 sequences had 16S similarities lower than 90% to database entries, suggesting that they represent previously undescribed species. In addition, three fungal species were also identified. Very different 16S rDNA DGGE banding profiles were obtained when replicate cv. Valencia leaf samples were cultured in BIOLOG EcoPlates for 4.5 days. All of these rDNA sequences had 97–100% similarity to those of known phyllosphere bacteria, but only two of them matched those identified by the culture independent DGGE analysis. Like other studied ecosystems, microbial phyllosphere communities therefore are more complex than previously thought, based on conventional culture-based methods.

p19ARF targets certain E2F species for degradation

p19ARF suppresses the growth of cells lacking p53 through an unknown mechanism. p19ARF was found to complex with transcription factors E2F1, -2, and -3. Levels of endogenous or ectopically expressed E2F1, -2, and -3, but not E2F6, were reduced after synthesis of p19ARF, through a mechanism involving increased turnover. p19ARF-induced degradation of E2F1 depended on a functional proteasome, and E2F1 was relocalized to nucleoli when coexpressed with p19ARF. Consistent with reduced levels of E2F1 and E2F3, the proliferation of cells defective for p53 function was suppressed by p19ARF, and the effect was partially reversed by ectopic overexpression of E2F1. These results suggest a broader role for p19ARF as a tumor suppressor, in which targeting of certain E2F species may cooperate with stimulation of the p53 pathway to counteract oncogenic growth signals.

Mapping of contact sites in complex formation between transducin and light-activated rhodopsin by covalent crosslinking: Use of a photoactivatable reagent

Interaction of light-activated rhodopsin with transducin (T) is the first event in visual signal transduction. We use covalent crosslinking approaches to map the contact sites in interaction between the two proteins. Here we use a photoactivatable reagent, N-[(2-pyridyldithio)-ethyl], 4-azido salicylamide. The reagent is attached to the SH group of cytoplasmic monocysteine rhodopsin mutants by a disulfide-exchange reaction with the pyridylthio group, and the derivatized rhodopsin then is complexed with T by illumination at λ >495 nm. Subsequent irradiation of the complex at λ310 nm generates covalent crosslinks between the two proteins. Crosslinking was demonstrated between T and a number of single cysteine rhodopsin mutants. However, sites of crosslinks were investigated in detail only between T and the rhodopsin mutant S240C (cytoplasmic loop V-VI). Crosslinking occurred predominantly with Tα. For identification of the sites of crosslinks in Tα, the strategy used involved: (i) derivatization of all of the free cysteines in the crosslinked proteins with N-ethylmaleimide; (ii) reduction of the disulfide bond linking the two proteins and isolation of all of the Tα species carrying the crosslinked moiety with a free SH group; (iii) adduct formation of the latter with the N-maleimide moiety of the reagent, maleimido-butyryl-biocytin, containing a biotinyl group; (iv) trypsin degradation of the resulting Tα derivatives and isolation of Tα peptides carrying maleimido-butyryl-biocytin by avidin-agarose chromatography; and (v) identification of the isolated peptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. We found that crosslinking occurred mainly to two C-terminal peptides in Tα containing the amino acid sequences 310–313 and 342–345.