Analyzing the function of a hox gene: an evolutionary approach

We present an evolutionary approach to dissecting conserved developmental mechanisms. We reason that important mechanisms for making the bodyplan will act early, to generate the major features of the body and that they will be conserved in evolution across many metazoa, and thus, that they will be available in very different animals. This led to our specific approach of microarrays to screen for very early conserved developmental regulators in parallel in an insect, Drosophila and a vertebrate, Xenopus. We screened for the earliest conserved targets of the ectopically expressed hox gene Hoxc6/Antennapedia in both species and followed these targets up, using in situ hybridization, in the Xenopus system. The results indicate that relatively few of the early Hox target genes are conserved: these are mainly involved in the specification of the antero-posterior body axis and in gastrulation.     

Site-directed mutagenesis reveals the importance of conserved charged residues for the transport activity of the PheP permease of Escherichia coli.

Site-directed mutagenesis has been used to identify a number of charged residues essential for the transport activity of the PheP protein. These residues are highly conserved in the cluster of amino acid transporters. However, some other conserved residues and a number of aromatic residues have been shown not to be essential for transport activity.     

Mutational analysis of the conserved domains of a T-region border repeat of Agrobacterium tumefaciens

Left-and right-border repeats, which surround the T-region, contain two conserved regions separated by 5 bp that are not conserved. At the onset of T-DNA processing virD-encoded proteins introduce a nick in the largest of these conserved regions (12 bp) at a specific position in the bottom strand between a guanine and thymine nucleotide [2, 33]. In this paper we describe the effect of several site-directed mutations in the right-border repeat on tumorigenicity of Agrobacterium in plants. Our data show that mutations introduced directly around the nick site do not seriously affect the tumorigenicity of Agrobacterium, whereas mutations in the right part of this 12 bp conserved region do so. Furthermore, it appeared that the second conserved region (5 bp) is also essential for border activity and that the distance between the two conserved regions is important to obtain optimal border activity.     

A model of the statistical power of comparative genome sequence analysis

Comparative genome sequence analysis is powerful, but sequencing genomes is expensive. It is desirable to be able to predict how many genomes are needed for comparative genomics, and at what evolutionary distances. Here I describe a simple mathematical model for the common problem of identifying conserved sequences. The model leads to some useful rules of thumb. For a given evolutionary distance, the number of comparative genomes needed for a constant level of statistical stringency in identifying conserved regions scales inversely with the size of the conserved feature to be detected. At short evolutionary distances, the number of comparative genomes required also scales inversely with distance. These scaling behaviors provide some intuition for future comparative genome sequencing needs, such as the proposed use of “phylogenetic shadowing” methods using closely related comparative genomes, and the feasibility of high-resolution detection of small conserved features.     

Significance in replication of the terminal nucleotides of the flavivirus genome

Point mutations that resulted in a substitution of the conserved 3'-penultimate cytidine in genomic RNA or the RNA negative strand of the self-amplifying replicon of the Flavivirus Kunjin virus completely blocked in vivo replication. Similarly, substitutions of the conserved 3'-terminal uridine in the RNA negative or positive strand completely blocked replication or caused much-reduced replication, respectively. The same preference for cytidine in the 3'-terminal dinucleotide was noted in reports of the in vitro activity of the RNA-dependent RNA polymerase (RdRp) for the other genera of Flaviviridae that also employ a double-stranded RNA (dsRNA) template to initiate asymmetric semiconservative RNA positive-strand synthesis. The Kunjin virus replicon results were interpreted in the context of a proposed model for initiation of RNA synthesis based on the solved crystal structure of the RdRp of phi6 bacteriophage, which also replicates efficiently using a dsRNA template with conserved 3'-penultimate cytidines and a 3'-terminal pyrimidine. A previously untested substitution of the conserved pentanucleotide at the top of the 3'-terminal stem-loop of all Flavivirus species also blocked detectable in vivo replication of the Kunjin virus replicon RNA.     

Computational approaches for the analysis of gene neighbourhoods in prokaryotic genomes

Gene order in prokaryotes is conserved to a much lesser extent than protein sequences. Only some operons, primarily those that encode physically interacting proteins, are conserved in all or most of the bacterial and archaeal genomes. Nevertheless, even the limited conservation of operon organisation that is observed provides valuable evolutionary and functional clues through multiple genome comparisons. With the rapid growth in the number and diversity of sequenced prokaryotic genomes, functional inferences for uncharacterized genes located in the same conserved gene neighborhood with well-studied genes are becoming increasingly important. In this review, we discuss various computational approaches for identification of conserved gene strings and construction of local alignments of gene orders in prokaryotic genomes.     

1H NMR investigation of the solution structure of substrate-free human heme oxygenase: comparison to the cyanide-inhibited, substrate-bound complex

1H NMR was used to investigate the molecular structure, and dynamic properties of soluble, recombinant, substrate-free human heme oxygenase (apohHO) on a comparative basis with similar studies on the substrate complex. Limited but crucial sequence-specific assignments identify five conserved secondary structural elements, and the detection of highly characteristic dipolar or H-bond interactions among these elements together with insignificant chemical shift differences confirm a strongly conserved folding topology of helices C-H relative to that of substrate complexes in either solution or the crystal. The correction of the chemical shifts for paramagnetic and porphyrin ring current influences in the paramagnetic substrate complex reveals that the strength of all but one of the numerous relatively robust H-bonds are conserved in apohHO, and similar ordered water molecules are located near these H-bond donors as observed in the substrate complexes. The unique and significant weakening of the Tyr(58) OH hydrogen bond to the catalytically critical Asp(140) carboxylate in apohHO is suggested to arise from the removal of the axial H-bond acceptor ligand rather than the loss of substrate. The interhelical positions of the conserved strong H-bonds argue for a structural role in maintaining a conserved structure for helices C-H upon loss of substrate. While the structure and H-bond network are largely conserved upon loss of substrate, the variably increased rate of NH lability dictates a significant loss of dynamic stability in the conserved structure, particularly near the distal helix F.     

Conservation of the MHC-like region throughout evolution

Identification of conserved regions between the genomes of distant species is a crucial step in the reconstruction of the genomic organization of their last common ancestor. Here we confirm for the first time with robust evidence, the existence of a region of conserved synteny between the human genome and the Drosophila genome. This evolutionarily conserved synteny involves the human MHC and paralogous regions, and we identified 19 conserved genes between these two species in a Drosophila genomic region of less than 2 Mb. The statistical analysis of the distribution of these 19 genes between the Drosophila and human genomes shows that it cannot be explained by chance. Our study constitutes a first step towards the reconstruction of the genome of Urbilateria (the ancestor of all bilaterian) and allows for a better understanding of the evolutionary history of our genome as well as other metazoan genomes.     

A comparative structural analysis of the ADF/cofilin family

Actin-depolymerizing factor (ADF) and cofilin define a family of actin-binding proteins essential for the rapid turnover of filamentous actin in vivo. Here we present the 2.0 A crystal structure of Arabidopsis thaliana ADF1 (AtADF1), the first plant crystal structure from the ADF/cofilin (AC) family. Superposition of the four AC isoform structures permits an accurate sequence alignment that differs from previously reported data for the location of vertebrate-specific inserts and reveals a contiguous, vertebrate-specific surface opposite the putative actin-binding surface. Extending the structure-based sequence alignment to include 30 additional isoforms indicates three major groups: vertebrates, plants, and "other eukaryotes." Within these groups, several structurally conserved residues that are not conserved throughout the entire AC family have been identified. Residues that are highly conserved among all isoforms tend to cluster around the tryptophan at position 90 and a structurally conserved kink in alpha-helix 3. Analysis of surface character shows the presence of a hydrophobic patch and a highly conserved acidic cluster, both of which include several residues previously implicated in actin binding.     

Sequence conservation in Ig-like domains: the role of highly conserved proline residues in the fibronectin type III superfamily

The role of conserved proline residues in fibronectin type III (fnIII) domains is investigated. Surprisingly, none of the standard set of explanations for residue conservation applies. The proline residues are not apparently conserved for function, or stability, or to nucleate folding, or to promote stabilising interactions across domain boundaries. However, when the most highly conserved proline residues are mutated to alanine there is an increase in the rate of aggregation of a fnIII double-module construct. The results suggest that proline residues may be conserved at domain-domain boundaries in fnIII domains to prevent aggregation in multi-modular proteins.     

Analysis of the interaction of FtsZ with itself, GTP, and FtsA.

The interaction of FtsZ with itself, GTP, and FtsA was examined by analyzing the sensitivity of FtsZ to proteolysis and by using the yeast two-hybrid system. The N-terminal conserved domain consisting of 320 amino acids bound GTP, and a central region of FtsZ, encompassing slightly more than half of the protein, was cross-linked to GTP. Site-directed mutagenesis revealed that none of six highly conserved aspartic acid and asparagine residues were required for GTP binding. These results indicate that the specificity determinants for GTP binding are different than those for the GTPase superfamily. The N-terminal conserved domain of FtsZ contained a site for self-interaction that is conserved between FtsZ proteins from distantly related bacterial species. FtsZ320, which was truncated at the end of the conserved domain, was a potent inhibitor of division although it expressed normal GTPase activity and could polymerize. FtsZ was also found to interact directly with FtsA, and this interaction could also be observed between these proteins from distantly related bacterial species.     

Comparative sequence analysis as a tool for studying the secondary structure of mRNAs.

Analysis of phylogenetically conserved secondary structure has been important in the development of models for the secondary structure of structural RNAs. In this paper, we apply this type of analysis to several families of informational RNAs to evaluate its usefulness in developing secondary structure models for mRNAs and mRNA precursors. We observed many conserved helices in all mRNA groups analyzed. Three criteria were used to identify potential helices which were not conserved solely because of coding sequence constraints, and may therefore be important for the structure and function of the RNA. These results suggest that this approach will be useful in deriving secondary structure models for informational RNAs when used in conjunction with other complementary techniques, and in designing experiments to determine the functional significance of conserved base pairing interactions.     

Co-conservation of rRNA tetraloop sequences and helix length suggests involvement of the tetraloops in higher-order interactions

Terminal loops containing four nucleotides (tetraloops) are common in structural RNAs, and they frequently conform to one of three sequence motifs, GNRA, UNCG, or CUUG. Here we compare available sequences and secondary structures for rRNAs from bacteria, and we show that helices capped by phylogenetically conserved GNRA loops display a strong tendency to be of conserved length. The simplest interpretation of this correlation is that the conserved GNRA loops are involved in higher-order interactions, intramolecular or intermolecular, resulting in a selective pressure for maintaining the lengths of these helices. A small number of conserved UNCG loops were also found to be associated with conserved length helices, consistent with the possibility that this type of tetraloop also takes part in higher-order interactions.     

Structural sequences are conserved in the genes coding for the alpha, alpha' and beta-subunits of the soybean 7S seed storage protein.

Cloned DNAs encoding four different proteins have been isolated from recombinant cDNA libraries constructed with Glycine max seed mRNAs. Two cloned DNAs code for the alpha and alpha'-subunits of the 7S seed storage protein (conglycinin). The other cloned cDNAs code for proteins which are synthesized in vitro as 68,000 d., 60,000 d. or 53,000 d. polypeptides. Hybrid selection experiments indicate that, under low stringency hybridization conditions, all four cDNAs hybridize with mRNAs for the alpha and alpha'-subunits and the 68,000 d., 60,000 d. and 53,000 d. in vitro translation products. Within three of the mRNA, there is a conserved sequence of 155 nucleotides which is responsible for this hybridization. The conserved nucleotides in the alpha and alpha'-subunit cDNAs and the 68,000 d. polypeptide cDNAs span both coding and noncoding sequences. The differences in the coding nucleotides outside the conserved region are extensive. This suggests that selective pressure to maintain the 155 conserved nucleotides has been influenced by the structure of the seed mRNA. RNA blot hybridizations demonstrate that mRNA encoding the other major subunit (beta) of the 7S seed storage protein also shares sequence homology with the conserved 155 nucleotide sequence of the alpha and alpha'-subunit mRNAs, but not with other coding sequences.     

Sequence and properties of the human KB cell and mouse L cell D-loop regions of mitochondrial DNA.

The sequences of the displacement-loop (D-loop) regions of mitochondrial DNA (mtDNA) from mouse L cells and human KB cells have been determined and provide physical maps to aid in the identification of sequences involved in the regulation of replication and expression of mammalian mtDNA. Both D-loop regions are bounded by the genes for tRNAPhe and tRNAPro. This region contains the most highly divergent sequences in mtDNA with the exceptions of three small conserved sequence blocks near the 5' ends of D-loop strands, a 225 nucleotide conserved sequence block in the center of the D-loop strand template region, and a short sequence associated with the 3' ends of D-loop strands. A sequence similar to that associated with the 3' termini of D-loop strands overlaps one of the conserved sequence blocks near the 5' ends of D-loop strands. The large, central conserved sequence probably does not code for a protein since no open reading frames are discretely conserved. Numerous symmetric sequences and potential secondary structures exist in these sequences, but none appear to be clearly conserved between species.