Regions Of High Homology Research Articles

Transcription, organization, and sequence of an auxin-regulated gene cluster in soybean.

We have characterized a soybean gene cluster that encodes a group of auxin-regulated RNAs (small auxin up RNAs). DNA sequencing of a portion of the locus reveals five homologous genes, spaced at intervals of about 1.25 kilobases and transcribed in alternate directions. At least three of the genes are transcriptionally regulated by auxin. An increase in the rate of transcription is detected 10 min after application of auxin to soybean elongating hypocotyl sections. Each of the genes contains an open reading frame that could encode a protein of 9 kilodaltons to 10.5 kilodaltons. Sequence comparisons among the five genes reveal several areas of high homology. Two regions of high homology begin about 250 base pairs upstream of the open reading frames and two regions of homology have been identified in sequences downstream of the open reading frames. One of the latter sequences occurs in the 3'-untranslated region of the RNAs. The other occurs far downstream, 618 base pairs to 741 base pairs from the stop codon. Conservation of these sequences among the five different genes suggests that they may be important for the regulation of expression of the genes.

Location of a single tRNA-His gene on the master chromosome of sunflower mitochondrial DNA

A highly purified tRNA fraction isolated from sunflower mitochondria was used to probe a restriction fragment library of sunflower mitochondrial DNA. Approximately 20 different clones were detected. The 0.2-kbp HindIII insert of recombinant plasmid pHD500 was sequenced revealing the presence of a tRNA-His gene with 97% similarity to the equivalent of maize mitochondria and chloroplast. The single copy gene appears to be located at least 30 kbp distant from regions of high homology with cpDNA in the mitochondrial genome. This observation and other evidence (analysis of flanking regions and hybridization to cpDNA) suggest that the gene probably does not belong to a chloroplast insertion. The possible expression of the tRNA-His gene is discussed.

First domain encoding sequence mediates human class II beta-chain gene cross-hybridization.

HLA class II molecules are surface heterodimers which are essential in the initiation of immune responses. The amount of polymorphism expressed by the different class II molecules is largely dependent on the polymorphic structure of their beta chains. Cross-hybridization between class II beta genes frequently hampered restriction fragment length polymorphism analysis of donor genomic DNA. In this report we show that the cross-hybridization between human class II beta genes is mediated by a region of high homology, rich in C and G residues, between the first domain encoding sequences of DP, DQ, and DR genes. The removal of the DNA segment containing this region from the fragments used as labeled probes against the corresponding fragments of the genes at other loci or against endonuclease digested genomic DNA completely eliminated or drastically reduced the cross-hybridization. Also, the RFLP patterns generated with the shortened probes were more informative and much simpler to interpret than were these generated with probes made from the original genes.

DNA sequence of the adenovirus type 41 hexon gene and predicted structure of the protein.

The gene for the major capsid protein (hexon) of human adenovirus type 41 (Ad41) has been isolated and the complete DNA sequence determined. Comparison of the predicted amino acid sequence with hexons from human Ad2 and Ad5 and bovine adenovirus type 3 reveals regions of high homology at the N and C termini separated by a central region of low homology. Fitting of the Ad41 hexon sequence to the known three-dimensional structure of the Ad2 hexon demonstrates that both hexons have a common architecture. Regions of the hexon which in the trimer constitute the pseudohexagonal base are highly conserved, with the major amino acid changes concentrated in the domains forming the triangular towers which represent the surface of the capsid. Changes in the Ad41 towers therefore permit the virus to present a unique surface to the environment while conservation of residues in the base maintains the integrity of hexon-hexon contacts. A striking difference is the absence in the Ad41 sequence of 32 amino acids which are present in the Ad2 sequence. In Ad2 this region is highly charged and may be responsible for pH-induced conformational changes within the virus capsid. The DNA sequence in the region surrounding the Ad41 hexon gene was also determined and revealed an open reading frame which appeared to code for the homologue of the Ad2-coded endoprotease. Comparison of the predicted amino acid sequences of the Ad41 and Ad2 proteins revealed a high degree of homology suggesting that this protein may have an important role in the infectious cycle of the virus.

Sequence and characterization of two auxin-regulated genes from soybean.

The auxin-regulated expression of two poly(A)+ mRNAs in soybean hypocotyl was demonstrated by cloning of the cDNAs and Northern blot hybridization analyses (Walker, J.C., and Key, J.L. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7185-7189). The corresponding genes, designated Aux28 and Aux22, have been isolated, and the cDNAs and genes have been sequenced. The Aux28 and Aux22 genes are present at one to two copies per haploid genome, contain four and two introns, and encode hydrophilic proteins of 26.8 and 21.5 kDa, respectively. Although the cDNAs were isolated independently and do not cross-hybridize under stringent hybridization conditions, the protein coding sequences of the two cDNAs have several colinear regions of high homology at the nucleic acid (77-80%) and the amino acid (80-100%) levels; together these regions constitute approximately a third of the protein coding sequences of the cDNAs. These data, together with genomic Southern blot hybridization analysis and hybrid-select translations of mRNAs homologous to the cDNAs, show that these genes belong to two related multigene families. We have identified two sequences, TGATAAAAG and GGCAGCATGCA, that occur at similar distances upstream of the transcription start site in each gene, and the spacing between these two elements is essentially identical in the two genes. The possible significance of these sequences is under evaluation.

The ras superfamily proteins

Several recent discoveries indicate that the ras genes, frequently activated to a transforming potential in some human tumours, belong to a large family that can be divided into three main branches: the first branch represented by the ras, ral and rap genes: the second branch, by the rho genes; and the third branch, by the rab genes. The C-terminal end of the encoded proteins always includes a cystein, which may become fatty-acylated, suggesting a sub-membrane localization. The ras superfamily proteins share four regions of high homology corresponding to the GTP binding site; however, even in these regions, significant differences are found, suggesting that the various proteins may possess slightly different biochemical properties. Recent reports show that some of these proteins play an essential role in the control of physical processes such as cell motility, membrane ruffling, endocytosis and exocytosis. Nevertheless, the characterization of the proteins directly interacting with the ras or ras-related gene-products will be required to precisely understand their function.

Connexin43: a protein from rat heart homologous to a gap junction protein from liver.

Northern blot analysis of rat heart mRNA probed with a cDNA coding for the principal polypeptide of rat liver gap junctions demonstrated a 3.0-kb band. This band was observed only after hybridization and washing using low stringency conditions; high stringency conditions abolished the hybridization. A rat heart cDNA library was screened with the same cDNA probe under the permissive hybridization conditions, and a single positive clone identified and purified. The clone contained a 220-bp insert, which showed 55% homology to the original cDNA probe near the 5' end. The 220-bp cDNA was used to rescreen a heart cDNA library under high stringency conditions, and three additional cDNAs that together spanned 2,768 bp were isolated. This composite cDNA contained a single 1,146-bp open reading frame coding for a predicted polypeptide of 382 amino acids with a molecular mass of 43,036 D. Northern analysis of various rat tissues using this heart cDNA as probe showed hybridization to 3.0-kb bands in RNA isolated from heart, ovary, uterus, kidney, and lens epithelium. Comparisons of the predicted amino acid sequences for the two gap junction proteins isolated from heart and liver showed two regions of high homology (58 and 42%), and other regions of little or no homology. A model is presented which indicates that the conserved sequences correspond to transmembrane and extracellular regions of the junctional molecules, while the nonconserved sequences correspond to cytoplasmic regions. Since it has been shown previously that the original cDNA isolated from liver recognizes mRNAs in stomach, kidney, and brain, and it is shown here that the cDNA isolated from heart recognizes mRNAs in ovary, uterus, lens epithelium, and kidney, a nomenclature is proposed which avoids categorization by organ of origin. In this nomenclature, the homologous proteins in gap junctions would be called connexins, each distinguished by its predicted molecular mass in kilodaltons. The gap junction protein isolated from liver would then be called connexin32; from heart, connexin43.

Genetic analysis of the H-2D region using a new intra-D-region recombinant mouse strain.

A rare D-region recombination event which gave rise to the B10.RQDB major histocompatibility complex haplotype has been examined to ascertain the nature of the crossover and to determine which class I genes are present in the new alignment of D-region genes. Serologic analysis have shown that the B10 . RQDB major histocompatibility complex recombinant mouse inherited the H-2Dd gene from the B10.T(6R) parental line and the H-2Db gene from the B10.A(2R) parental line, representing the first example of an intra-D-region crossover resulting from an intercross. Previous molecular genetic analyses of the d and b haplotypes revealed structural diversity in the organization of their D-region gene clusters. Hence, the D region is comprised of five class I genes in the d haplotype and only one in the b haplotype. Because allelic relationships among the various D-region genes are not defined, either a homologous or nonhomologous alignment of genes has generated the RQDB crossover. Therefore, the possibility that all three D-region antigen-presenting molecules (Dd, Ld, and Db) might be encoded by the RQDB haplotype was examined. Fluorescence-activated cell sorter and cytotoxic T lymphocyte analyses revealed no detectable levels of H-2Ld cell-surface expression, confirming earlier studies with antibody-mediated cytotoxicity and immunoprecipitation. Southern blot analysis localized the recombination point to within a 1-kb region at the centromeric end of the H-2Ld gene on the B10 . T(6R) chromosome in a region of high homology to the H-2Db gene on the B10 . A(2R) chromosome. Together, these studies define the D region of the RQDB haplotype as containing the five class I genes: Dd, D2d, D3d, D4d, and Db. In addition to providing insight into rare recombination events in the D region, the B10.RQDB mouse should be a useful tool for exploring the function of D-region genes.

Topological mapping of complement component C9 by recombinant DNA techniques suggests a novel mechanism for its insertion into target membranes.

cDNA molecules coding for mouse and trout C9 have been isolated and the derived amino acid sequences compared with that of human C9. Regions of high homology between the closely related species (mouse and human) correlate with putative domains in the protein structure supporting a model of C9 having five globular domains. Comparison between the more distant species (trout and human) suggests regions of particular importance to C9 structure and function. In addition the three related sequences allow the secondary structure to be predicted with more confidence and we have tested the prediction by mapping surface features of the protein. Reported here is a recombinant DNA approach to fine mapping of antibody epitopes. Two of the putative domains of C9 are connected by a stretch of about 40 amino acid residues in which features characteristic of individual conformational forms of C9 are concentrated. We suggest that this region might act as a hinge allowing the rearrangement of globular domains necessary for membrane insertion. In the membrane inserting domain one highly conserved sequence has the potential to form an amphipathic alpha-helix once it is buried in the lipid bilayer. These features suggest a novel mechanism for the irreversible, post-translational insertion of C9 into target membranes.

The receptor-binding sequence of urokinase. A biological function for the growth-factor module of proteases.

Previous studies have shown that the region of human urokinase-type plasminogen activator (uPA) responsible for receptor binding resides in the amino-terminal fragment (ATF, residues 1-135) (Stoppelli, M.P., Corti, A., Soffientini, A., Cassani, G., Blasi, F., and Assoian, R.K. (1985) Proc. Natl. Acad. Sci. U.S. A. 82, 4939-4943). The area within ATF responsible for specific receptor binding has now been identified by the ability of different synthetic peptides corresponding to different regions of the amino terminus of uPA to inhibit receptor binding of 125I-labeled ATF. A peptide corresponding to human [Ala19]uPA-(12-32) resulted in 50% inhibition of ATF binding at 100 nM. Peptides uPA-(18-32) and [Ala13]uPA-(9-20) inhibit at 100 and 2000 microM, respectively. The human peptide uPA-(1-14) and the mouse peptide [Ala20]uPA-(13-33) have no effect on ATF receptor binding. This region of uPA is referred to as the growth factor module since it shares partial amino acid sequence homology (residues 14-33) to epidermal growth factor (EGF). Furthermore, this region of EGF is responsible for binding of EGF to its receptor (Komoriya, A. Hortsch, M., Meyers, C., Smith, M., Kanety, H., and Schlessinger, J. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1351-1355). However, EGF does not inhibit ATF receptor binding. Comparison of the sequences responsible for receptor binding of uPA and EGF indicate that the region of highest homology is between residues 13-19 and 14-20 of human uPA and EGF, respectively. In addition, there is a conservation of the spacings of four cysteines in this module whereas there is no homology between residues 20-30 and 21-33 of uPA and EGF. Thus, residues 20-30 of uPA apparently confer receptor binding specificity, and residues 13-19 provide the proper conformation to the adjacent binding region.

The nontranscribed chicken calmodulin pseudogene cross-hybridizes with mRNA from the slow-muscle troponin C gene.

A chicken calmodulin pseudogene with no introns was previously shown to hybridize under stringent conditions with an mRNA species present in skeletal and cardiac muscles, yet it would not hybridize to calmodulin mRNA (J. P. Stein, R. P. Munjaal, L. Lagace', E. C. Lai, B. W. O'Malley, and A. R. Means, Proc. Natl. Acad. Sci. USA 80:6485-6489, 1983). Using the pseudogene as a probe, we isolated a full-length cDNA corresponding to this mRNA from a chicken breast muscle library and showed by sequence analysis that it encodes slow-muscle troponin C and not the pseudogene product. Hybridization between the calmodulin pseudogene and slow-muscle troponin C cDNA is due to a short region of high homology in those nucleotides that encode helices B and C of troponin C and calmodulin. Genomic Southern analysis showed the calmodulin pseudogene and the gene for slow-muscle troponin C to exist as distinct single copies.

The nontranscribed chicken calmodulin pseudogene cross-hybridizes with mRNA from the slow-muscle troponin C gene.

A chicken calmodulin pseudogene with no introns was previously shown to hybridize under stringent conditions with an mRNA species present in skeletal and cardiac muscles, yet it would not hybridize to calmodulin mRNA (J. P. Stein, R. P. Munjaal, L. Lagace', E. C. Lai, B. W. O'Malley, and A. R. Means, Proc. Natl. Acad. Sci. USA 80:6485-6489, 1983). Using the pseudogene as a probe, we isolated a full-length cDNA corresponding to this mRNA from a chicken breast muscle library and showed by sequence analysis that it encodes slow-muscle troponin C and not the pseudogene product. Hybridization between the calmodulin pseudogene and slow-muscle troponin C cDNA is due to a short region of high homology in those nucleotides that encode helices B and C of troponin C and calmodulin. Genomic Southern analysis showed the calmodulin pseudogene and the gene for slow-muscle troponin C to exist as distinct single copies.

Structural Organization and Regulation of the Chicken Estrogen Receptor

We have cloned the chicken estrogen receptor (ER) from a chicken oviduct lambda gt11 library using the human ER cDNA sequence. This chicken ER sequence is virtually identical to the recently published sequence. One noteable difference is an amino acid change from glutamine to arginine located toward the central region of the sequence. The size of the ER protein predicted from the 589 amino acids is approximately 66,000 which fits well with the range of molecular weights previously published for the calf uterine and human ER (65,000-70,000). We observed the size of the chicken ER mRNA to be approximately 7.8 kilobases which is in agreement with the previously published size of 7.5 kilobases. In vivo secondary stimulation of chicken oviduct total RNA with diethylstilbestrol does not induce chicken ER mRNA. A time course following the chicken ER mRNA levels after secondary stimulation with diethylstilbestrol indicated a decrease in mRNA levels 8 h after DES administration. A similar study was performed using progesterone for the secondary stimulation. An increase in the chicken ER mRNA levels was observed 24 h after stimulation with progesterone. Two regions of very high homology were delineated by analyzing the sequence of this chicken ER cDNA and comparing it to the sequences of the human ER, human glucocorticoid, and chicken progesterone receptors and the P75-erbA fusion product of the avian erythroblastosis virus. The first concensus region is 72 amino acids in length and the second region of high homology is 62 amino acids long. Detailed comparisons of these regions for the steroid hormone receptors and v-erb A are presented. Possible functions for the individual regions of high homology are discussed.

Epidermal growth factor receptor occupancy inhibits vaccinia virus infection.

Vaccinia virus encodes VGF, an early protein of relative molecular mass 19,000 (19K) which, from amino-acid residues 45 to 85, is homologous in 19 residues to epidermal growth factor (EGF), and transforming growth factor-alpha (TGF-alpha). The conserved sequence includes a region of high homology (6 out of 10 amino acids) from residues 71 to 80, corresponding to the third disulphide loop of both EGF and TGF-alpha. This region has recently been shown to contain a binding region of TGF-alpha for the EGF receptor, and this raises the question of whether vaccinia virus utilizes the EGF receptor in order to bind to and infect cells. We now show that occupancy of the EGF receptor inhibits vaccinia virus infection. Inhibition is observed in a dose-dependent fashion by pre-treatment with either EGF or synthetic decapeptide antagonists of EGF's mitogenic activity which correspond to the sequence of the third disulphide loop of VGF or TGF-alpha. The relative ability of the peptides to inhibit vaccinia virus infection parallels their binding affinity to the EGF receptor.

Cloning and sequencing of the adenylate kinase gene (adk) of Escherichia coli.

Adenylate kinase, the product of the adk locus in Escherichia coli K12, catalyzes the conversion of AMP and ATP to two molecules of ADP. The gene has been cloned by complementation of an adk temperature sensitive mutation. The DNA sequence of the complete coding region and of 5'- and 3'-untranslated regions were determined. The resulting protein sequence was found to contain several regions of high homology with cytosolic adenylate kinase of pig muscle (AK1), whose three-dimensional structure has been determined. The most significant of the amino acid exchanges is the replacement of histidine 36 with glutamine. This residue is believed to play a role in catalysis through metal ion binding. The codon usage pattern and the determination of adenylate kinase molecules per cell shows that the enzyme is one of the more abundant soluble proteins of the bacterial cells.

Herpes simplex virus types 1 and 2 homology in the region between 0.58 and 0.68 map units.

The homology between herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2, respectively) DNA between 0.58 and 0.674 map units was compared by Southern and dot blot analysis with DNA of one type of virus as a hybridization probe against the other type. Regions of high homology were interspersed with regions of detectably lower homology. However, only one region (between 0.647 and 0.653 map units) contained few or no homologous sequences. In situ RNA blot hybridization demonstrated that the mRNA species transcribed in the right-hand portion of the region are homologous between HSV-1 and HSV-2, as was previously found for the left-hand portion. A 2.7-kilobase HSV-2 transcript in the right-hand portion of the studied region was clearly that encoding HSV-2 glycoprotein C. Comparative nucleotide sequence analysis of specific regions demonstrated that homologous translational reading frames could be identified in the virus types. This analysis also demonstrated that homology could be abruptly lost outside such reading frames. Comparison of regions of homology with published HSV-1 transcription maps suggests that there can also be large divergence within translational reading frames. Some, but not complete, sequence homology was seen in the putative promoter sequence for the 730-base HSV-1 mRNA mapping to the right of glycoprotein C and the corresponding HSV-2 DNA. This suggests that the rather strict conservation of promoter sequences between homologous HSV-1 and HSV-2 transcripts seen in other regions of the genome may not be a necessary feature between these virus types.

Sequence homology between polyoma virus, simian virus 40, and a papilloma-producing virus from a syrian hamster: Evidences for highly conserved sequences

Sequence homology between the genomes of a hamster papovavirus (HaPV), polyoma virus (Py), and Simian virus 40 (SV40) has been studied by filter hybridization and electron microscopy under conditions of varying stringency. Hybrids between the HaPV and SV40 DNAs could be demonstrated only under nonstringent conditions. The region of highest homology was mapped in the early region of the SV40 genome. Extensive homology was detected between the genomes of HaPV and Py under stringent hybridization conditions, indicating at least 80% base matching in the regions of strongest sequence homology. These sequences were localized within both the early region and the late region of the Py genome. The homologous DNA segments mapped in the Py and the SV40 genomes are among the most strongly conserved regions in the polyoma (miopapova)-virus group.

Only site-directed antibodies reactive with the highly conserved src-homologous region of the v-abl protein neutralize kinase activity.

Antisera specific for six regions of the v- abl protein were used to serologically characterize the Abelson murine leukemia virus tyrosine kinase. Chemically synthesized peptides corresponding to the predicted v- abl protein sequence and larger regions of the v- abl protein expressed as fusion proteins in bacteria were used as immunogens. The specificity of each antiserum was confirmed by immunoprecipitation analysis with defined deletion mutants of Abelson murine leukemia virus. Several of these v- abl -specific antisera display much higher titers and avidities than serum harvested from mice bearing Abelson murine leukemia virus-induced tumors, previously the only source of anti- abl -specific serum. Two antisera were found to block the in vitro autophosphorylation of the v- abl protein as well as its ability to phosphorylate a peptide substrate. Examination of the sites against which the kinase-blocking antisera were prepared revealed that both are in close proximity to the in vivo sites of tyrosine phosphorylation, which fall within the region of high homology with v-src and other tyrosine kinases. Antisera directed against other regions of v- abl did not inhibit kinase activity.

Restriction map and southern analysis of the bean golden mosaic virus genome

A restriction map showing 5′–3′ polarity for two species of circular single-stranded (ss) DNA from bean golden mosaic virus (BGMV) is presented. The two viral single-stranded DNAs, which are very similar in size, were separated by prolonged agarose gel electrophoresis at low voltage. Hybridization analysis using probes made from these separated DNAs shows they are largely dissimilar in nucleotide sequence except for a common region of high homology which has been mapped to a unique location on each species. Molecular clones of BGMV DNA from restriction fragments comprising all of one of the two DNAs and part of the other were used to confirm the restriction and hybridization analysis. These results provide further evidence consistent with the hypothesis that BGMV and other geminiviruses possess divided genomes with two components of nearly equal size.

Nucleotide sequence of Escherichia coli pabA and its evolutionary relationship to trp (G) D

We have determined the entire nucleotide sequence of Escherichia coli pabA. A comparison of the nucleotide and amino acid sequences of pabA and trp(G) D reveals extensive homology, suggesting that these two genes arose from a common ancestor. pabA and trp(G) D are 44% homologous at the amino acid level and 53% homologous at the nucleotide sequence level. The nucleotide sequences can be divided into regions of high homology, in which most nucleotide changes occur in the third position of codons and do not effect the amino acid sequence, and regions which show almost no DNA homology. Divergence in these non-homologous regions appears to have resulted from single-base substitutions as well as the rearrangement of small regions of DNA by inversion, deletion and duplication.