A gal region mutant that requires cAMP for growth on galactose in an adenyl cyclase negative (cya delta) background.

Summary Strains ofEscherichia coli K12 that contain a deletion of the adenyl cyclase gene (cya), required for the synthesis of cyclic adenosine-3; 5 monophosphate (cAMP), grow on galactose-containing minimal medium. A mutant was isolated that grows on this medium only if cAMP is added. The mutation (designatedgalP20) is linked to thegal operon region as determined by both generalized transduction with bacteriophage P1 and specialized transduction with bacteriophage . Studies withgalP20 cya strains as well asgal (deletions of thegal operon)cya strains indicate that synthesis of the physiologically important transport mechanism for galactose (galactose permease) requires either cAMP or a function missing from both thegal strains and thegalP20 strain.     

Chromosomal location and expression of the single-copy gene encoding high-mobility-group protein HMG-I/Y in Arabidopsis thaliana

A cDNA encoding the HMG-I/Y protein from Arabidopsis thaliana has been isolated and characterised by nucleotide sequencing. The 903 bp cDNA contains a 612 bp open reading frame encoding a protein of 204 amino acid residues showing homology to HMG-I/Y proteins from other plant species. The protein contains four copies of the AT-hook motif which is involved in binding A/T-rich DNA. Southern blotting showed that the HMG-I/Y gene was present in a single copy in the Arabidopsis genome. The gene was localised to the top of chromosome 1 by RFLP analysis of F₈ recombinant inbred lines. Northern blotting showed that the gene was expressed in all organs examined, with the highest expression in flowers and developing siliques.     

Studies of platelets with heavy metal impregnation techniques

Summary Vaious methods of heavy metal impregnations were performed on human platelets. The optimal technique consisted of glutaraldehyde fixation, incubation in warm uranyl acetate at a pH of 3.5, followed by a double solution of lead and copper, and finally overnight immersion in cold osmium tetroxide. Semi-thin sections, viewed at 90 kV, revealed three types of platelets: (1) reticular cells, with a prominent tubular network and very dark granules in a pale cytoplasm; (2) dark cells, with an electron-dense cytoplasm; and (3) pale cells, with microvesicles and non-staining granules. Pre-treatments with EGTA, aspirin and various platelet activators altered the appearances and proportions of the three cell types.A cell-partitioning two-phase polymer system showed that the sub-grouping is related to surface membrane properties, the cells retained in the top phase being exclusively type 2 dark cells.The changes in cell type distribution produced by activation show that metal impregnation may be a useful method for studying structure-function correlations in platelets.     

An unusual arrangement of pur and lpx genes in the photosynthetic purple sulfur bacterium Allochromatium vinosum

The nucleotide sequence of a 1634 bp DNA fragment from the photosynthetic purple sulfur bacterium Allochromatium vinosum contains one complete and two partial open reading frames. Sequence comparisons to genes from other organisms suggest that this A. vinosum DNA fragment contains, starting from the 5 end, the following: (1) 234 bp at the 3 end of the A. vinosum purH gene, coding for 78 amino acids at the C-terminus of the bi-functional 5-phosphoribosyl-5-aminoimidazole-4-carboxamide formyltransferase/IMP cyclohydrolase (EC 2.1.2.3), an enzyme involved in de novo purine biosynthesis; (2) 777 bp of the A. vinosum lpxA gene, coding for all 259 amino acids of the UDP-N-acetylglucosamine-O-acyltransferase, an enzyme involved in lipid A biosynthesis; and (3) 567 bp at the 5 end of the A. vinosum purD gene, coding for 189 amino acids at the N-terminus of 5-phosphoribosyl glycinamide synthetase (EC 6.3.4.13), a second enzyme involved in de novo purine biosynthesis. The presence of a gene coding for an enzyme involved in lipid A biosynthesis between two genes coding for enzymes of the de novo purine biosynthesis pathway represents a unique arrangement of these genes.     

Sequence analysis of linked maize 22 kDa α-zein genes

We have determined the nucleotide sequence of a 7343 bp zein genomic clone (gZ22.8H3) from the maize inbred W64A. Computer-aided analysis of the DNA sequence revealed two contiguous 22 kDa -zein genes. The 5 gene (gZ22.8) encodes a complete polypeptide and contains putative regulatory sequences in both the 5 and 3 flanking regions that are typical of zein genes. In contrast, the 3 gene (gZ22.8) appears to be a pseudogene, because it contains numerous insertions and deletions that would prevent translation of the mRNA. Alignment of the 5 and 3 flanking sequences of both genes indicated that they resulted from a 3.3 kb DNA duplication event.     

The Correcting Role of Autonomous 3" →="" 5"="" exonucleases="" contained="" in="" mammalian="" multienzyme="" dna="" polymerase="" complexes"="

A study was made of the correcting role of autonomous 3" 5" exonucleases (AE) contained in multienzyme DNA polymerase complexes of rat hepatocytes or calf thymocytes. DNA was synthesized on phage X174 amber3 or M13mp2 primer–templates, and used to transfect Escherichia coli spheroplasts. Frequencies were estimated for direct and reverse mutations resulting from mistakes made in the course of in vitro DNA synthesis. The error rate of the hepatocyte complex was estimated at 3·10^–6 with equimolar dNTP, and increased tenfold when proteins accounting for 70% of the total 3" 5" exonuclease activity of the complex were removed. The fidelity of DNA synthesis was completely restored in the presence of exogenous AE ( subunit of E. coli DNA polymerase III). Nuclear (Pol _n) and cytosolic (Pol _c) forms of DNA polymerase were isolated from calf thymocytes. The former was shown to contain an AE (TREX2) absent from the latter. As compared with Pol _c, Pol _n had a 20-fold higher exo/pol ratio and allowed 4–5 times higher fidelity of DNA synthesis. The error rate of DNA polymerase complexes changed when dNTP were used in nonequimolar amounts.     

The linkage of Hb Valletta [α2β287(F3)Thr→Pro] and Hb F-Malta-I [α2 Gγ2117(G19)His→Arg] in the Maltese population

Summary We have identified a new stable abnormal hemoglobin called Hb Valletta, which is characterized by a ThrPro substitution at position 87 of the chain. This mutation was found to be linked to that of the chain variant Hb F-Malta-I with a HisArg mutation at position 117 of the ^G chain. Both variants were detected in the blood samples of 34 Maltese and two Italian new-born babies with isoelectrofocusing and reversed phase high performance liquid chromatography. Similar analyses of cord blood from 388 additional Maltese newborns failed to identify either one of these two variants. Additional analyses of 353 Maltese adults (including 39 -thalassemia heterozygotes) resulted in the detection of two adult Hb Valletta heterozygotes. Dot-blot hybridization analyses of amplified DNA with a probe specific for the ^G-F-Malta-I variant showed that both also carried that mutation. These results show close linkage of the mutant forms of the ^G- and -globin genes, 27–28 kb apart, and a failure to identify chromosomes with either the Hb F-Malta-I mutation alone or with the Hb Valletta mutation alone, indicating a low recombination frequency.     

A Maximum Likelihood Method for Detecting Functional Divergence at Individual Codon Sites, with Application to Gene Family Evolution

The tailoring of existing genetic systems to new uses is called genetic co-option. Mechanisms of genetic co-option have been difficult to study because of difficulties in identifying functionally important changes. One way to study genetic co-option in protein-coding genes is to identify those amino acid sites that have experienced changes in selective pressure following a genetic co-option event. In this paper we present a maximum likelihood method useful for measuring divergent selective pressures and identifying the amino acid sites affected by divergent selection. The method is based on a codon model of evolution and uses the nonsynonymous-to-synonymous rate ratio () as a measure of selection on the protein, with =1, <1, and >1 indicating neutral evolution, purifying selection, and positive selection, respectively. The model allows variation in among sites, with a fraction of sites evolving under divergent selective pressures. Divergent selection is indicated by different s between clades, such as between paralogous clades of a gene family. We applied the codon model to duplication followed by functional divergence of (i) the and globin genes and (ii) the eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN) genes. In both cases likelihood ratio tests suggested the presence of sites evolving under divergent selective pressures. Results of the and globin analysis suggested that divergent selective pressures might be a consequence of a weakened relationship between fetal hemoglobin and 2,3-diphosphoglycerate. We suggest that empirical Bayesian identification of sites evolving under divergent selective pressures, combined with structural and functional information, can provide a valuable framework for identifying and studying mechanisms of genetic co-option. Limitations of the new method are discussed.     

Genetic analysis of gliadin-encoding genes reveals gene clusters as well as single remote genes

Summary Analysis of F₂ grains from two different crosses has revealed a complex organization of the family of gliadin-coding genes located on chromosomes of the first homoeological group in hexaploid wheat. Chromosome 1A of variety Bezenchukskaya 98 was found to carry at least five gliadin-coding genes of which three genes form a cluster controlling the synthesis of the GLD1A1 block. Two additional genes are located on the both sides of this cluster and recombine with it at frequencies of 5±1.3% and 13±2.9%. Gliadinencoding genes recombining with the main clusters were also found on chromosomes 1B and 1A in the Bezenchukskaya 98 and Saratovskaya 210 varieties, respectively. In Chinese Spring, widely used in genetic studies, we discovered a recombination between genes located on chromosome 1A and controlling the synthesis of - and -gliadins. Varieties and biotypes of one variety may differ by the presence or absence of such selfish (not included in clusters) gliadin components. The similarity of organization of prolamine-coding genes on chromosomes in different cereals is considered.     

Molecular cloning of the T4 genome: organization and expression of the tail fiber gene cluster 34--38

Summary T4 derivatives that carry T4 tail fiber genes 34–38 have been isolated and characterized by genetic, structural and functional analysis. 32 T4 recombinants were identified by a marker rescue screen of 310 T4 clones generated by restriction of partial cytosine-containing T4 DNA with either HindIII or EcoRI and ligation into appropriately cleaved vectors. These tests defined 15 recombinant classes with respect to the contiguous stretches of genome recovered. Restriction enzyme structural analysis identified 7 HindIII fragments and 7 EcoRI fragments, established a restriction map covering about 11 kb, and indicated the orientation of the DNA inserts within the vectors. The cloned tail fiber genes are expressed efficiently from promoters and complement in vivo T4 phage carrying amber mutations in the tail fiber genes. Polypeptides corresponding to gp34-gp38 have been detected by SDS polyacrylamide gel electrophoresis of ³⁵S-labeled extracts of appropriate T4 recombinant infected UV-treated host cells. The genetic, structural and functional maps of the T4 tail fiber gene cluster have been correlated, and provide a rational approach to genetically directed DNA sequence analysis of genes 34–38 and their mutant variants that affect the assembly, structure and function of the tail fibers.     

Defective prophages of bacteriophage Mu

Summary A method is described for the isolation of thermoinducible defective Mu lysogens. Four of these defective lysogens were studied more extensively. By marker-rescue experiments it was shown that the strain harbouring the smallest defective prophage contains the immunity gene cts and the genes A and B; the strain with the largest defective prophage still contains all the known essential genes of Mu, A to S (see Fig. 1).After induction at 43° C all the defective lysogens are killed, whereas no lysis occurs.Although in all the thermoinducible defective lysogens the A and B gene products could be demonstrated by complementation, these gene products are not responsible for the killing of the host, suggesting the presence of another unknown early gene product of Mu. The level of complementation of a mutation in gene A is reduced by the presence in the cell of another defective Mu prophage containing the G part of Mu. This effect on A gene complementation is markedly enhanced when the defective prophage, containing the G part, is located on an episome instead of on the chromosome. Complementation of late genes by a defective prophage located on the chromosome, is extremely low or undetectable. A stimulation of complementation by a factor of 10 to 40 was found when the same defective prophage was situated on a F factor. A possible explanation for this episome effect will be discussed.     

Diallel analysis of tiller number at different growth stages in rice (Oryza sativa L.)

Summary The genetics of tiller number at different development stages in rice was studied in a 6 x 6 diallel cross set involving six indica rice varieties in which a large difference of phenotypes existed. Estimates of genetic parameters following Hayman's method showed significant additive and nonadditive gene action, and the latter appeared to be solely due to dominance. High tillering ability was inherited as a partial dominant character conditioned by two or more blocks of genes, and var H1459 had more dominant genes, whereas Zhu-Yun-Luo had more recessive ones. Combining ability analysis by Griffing's method also indicated significance of both additive and nonadditive effects, but the former was more important than the latter. H1459 and Bi-Yu-Zao-Luo were good general combiners, and their hybrids H1459 x Le-Yi and Zhu-Yun-Luo x Bi-Yu-Zao-Luo were better crosses for combining an optimum tiller number with high percentage of productive tiller. An identical polygenic system appeared to be responsible for the genetic control of both tiller number at the different growth stages and the terminal character, productive tiller number. However, with growth of the plant, relative contributions of nonadditive gene action and environmental factors to the variation decreased, while those of additive gene action increased. Selection for high tillering ability, therefore, should be exercised at peak tillering or later stages.     

Linkage of the β-Like ω-Globin Gene to α-Like Globin Genes in an Australian Marsupial Supports the Chromosome Duplication Model for Separation of Globin Gene Clusters

The structure, function, and evolutionary history of globin genes have been the subject of extensive investigation over a period of more than 40 years, yet new globin genes with highly specialized functions are still being discovered and much remains uncertain about their evolutionary history. Here we investigate the molecular evolution of the -globin gene family in a marsupial species, the tammar wallaby, Macropus eugenii. We report the complete DNA sequences of two -like globin genes and show by phylogenetic analyses that one of these genes is orthologous to embryonically expressed -globin genes of marsupials and eutherians and the other is orthologous to adult expressed -globin genes of marsupials and eutherians. We show that the tammar wallaby contains a third functional -like globin gene, -globin, which forms part of the -globin gene cluster. The position of -globin on the 3 side of the -globin cluster and its ancient phylogenetic history fit the criteria, originally proposed by Jeffreys et al. (1980), of a fossil -globin gene and suggest that an ancient chromosome or genome duplication preceded the evolution of unlinked clusters of - and -globin genes in mammals and avians. In eutherian mammals, such as humans and mice, -globin has been silenced or translocated away from the -globin locus, while in marsupials -globin is coordinately expressed with the adult -globin gene just prior to birth to produce a functional hemoglobin (_{2 2}).     

The chromosomal distribution of repetitive DNA sequences within the human β globin gene cluster

Summary The chromosomal distribution and degree of repetitiveness of the sequences within the human globin gene cluster has been studied by in situ hybridisation. A genomic recombinant, H\G1, which contains 15.9 thousand base pairs (kb) of DNA inserted into charon 4A, was used as a template for the [³H] complementary RNA used for hybridisation. The inserted DNA contains sequences 4.7 kb to the 5 side of the gene and continues through the and genes to a site 2.8 kb to the 3 side of the gene. Two highly repetitive sequences, which are distributed evenly over all the chromosomes, have been identified within this DNA. Another somewhat less repetitive sequence has been identified between the and genes.     

Molecular characterization of two types of 22 kilodalton α-zein genes in a gene cluster in maize

Summary Five genes of the -zein subfamily four (SF4) are located in a 56 kb genomic region of the maize inbred line W22. Their nucleotide and deduced amino acid sequences have been determined. The sequences define two types of -zein SF4 genes — type 1 (T1) and type 2 (T2). The single T1 -zein SF4 gene codes for an -zein protein with a M_r of about 22 000. This is the first -zein SF4 gene sequenced that contains no early in-frame stop codons in its coding sequence. The four T2 -zein SF4 genes in this cluster contain one or two early in-frame stop codons. In addition, our T1 and T2 genes differ markedly in the base sequences of their distal 5 non-translated flanking regions. The nucleotide and the deduced amino acid sequences of these two types of -zein SF4 genes are similar ( > 90 %) to one another and to all known -zein SF4 genes and cDNAs. Of the known W22 -zein SF4 genes, only one in six does not contain an early in-frame stop codon. If the number of -zein SF4 genes is 15–20, then we estimate that only about 4 of the W22 -zein SF4 genes are without in-frame early stop codons.     

A highly conserved α-tubulin sequence from Prunus amygdalus

The sequence of an -tubulin from Prunus amygdalus has been obtained by cDNA cloning. When this sequence is compared to that of the Tub1 gene from maize it shows a very high degree of similarity, much higher than any of the -tubulin sequences reported so far from plants. The expression of this gene is high in the stages of seed development where a high divisional activity is present. It is preferentially expressed in the radicular tissues as it is gene Tub1 in maize. Southern analysis indicates that this gene may from a subfamily of -tubulin genes having similar sequence and tissue specificity and existing at least in maize and in Prunus.     

Structural analysis of a maize gene coding for glutathione-S-transferase involved in herbicide detoxification

Summary We have used the cDNA clone encoding maize glutathione-S-transferase (GST I) to isolate a genomic DNA clone containing the complete GST I gene. Nucleotide sequence analysis of the cDNA and genomic clones has yielded a complete amino acid sequence for maize GST I and provided the exon-intron map of its gene. The mRNA homologous sequences in the maize GST I gene consist of a 107 bp 5 untranslated region, a 642 bp coding region and 340 bp of the 3 untranslated region. They are divided into three exons by two introns which interrupt the coding region. The 5 untranslated spacer contains an unusual sequence of pentamer AGAGG repeated seven times. The inbred maize line (Missouri 17) contains a single gene for GST I, whereas the hybrid line (3780A) contains two genes. Nucleotide sequence analysis of the primer extended cDNA products reveals that the 5 untranslated regions of the two genes in the hybrid 3780A are identical except for a 6 bp internal deletion (or insertion). The amino acid sequence of maize GST I shares no apparent sequence homology with the published sequences of animal GST's and represents the first published sequence of a plant GST. re]19850813 ac]19851126     

Classification and characterization of the rice α-amylase multigene family

To establish the size and organization of the rice -amylase multigene family, we have isolated 30 -amylase clones from three independent genomic libraries. Partial characterization of these clones indicates that they fall into 5 hybridization groups containing a total of 10 genes. Two clones belonging to the Group 3 hybridization class have more than one gene per cloned fragment. The nucleotide sequence of one clone from Group 1, OSg2, was determined and compared to other known cereal -amylase sequences revealing that OSg2 is the genomic analog of the rice cDNA clone, pOS103. The rice -amylase genes in Group 1 are analogous to the -Amy1 genes in barley and wheat. OSg2 contains sequence motifs common to most actively transcribed genes in plants. Two consensus sequences, TAACA _G ^A A and TATCCAT, were found in the 5 flanking regions of -amylase genes of rice, barley and wheat. The former sequence may be specific to -amylase gene while the latter sequence may be related to a CATC box found in many plant genes. Another sequence called the pyrimidine box ( _T ^C CTTTT _T ^C ) was found in the -amylase genes as well as other genes regulated by gibberellic acid (GA). Comparisons based on amino acid sequence alignment revealed that the multigene families in rice, barley and wheat shared a common ancestor which contained three introns. Some of the descendants of the progenitor -amylase gene appear to have lost the middle intron while others maintain all three introns.     

Resistance gene analogues identified through the NBS-profiling method map close to major genes and QTL for disease resistance in apple

We used a new method called nucleotide-binding site (NBS) profiling to identify and map resistance gene analogues (RGAs) in apple. This method simultaneously allows the amplification and the mapping of genetic markers anchored in the conserved NBS-encoding domain of plant disease resistance genes. Ninety-four individuals belonging to an F₁ progeny derived from a cross between the apple cultivars Discovery and TN10-8 were studied. Two degenerate primers designed from the highly conserved P-loop motif within the NBS domain were used together with adapter primers. Forty-three markers generated with NBS profiling could be mapped in this progeny. After sequencing, 23 markers were identified as RGAs, based on their homologies with known resistance genes or NBS/leucine-rich-repeat-like genes. Markers were mapped on 10 of the 17 linkage groups of the apple genetic map used. Most of these markers were organized in clusters. Twenty-five markers mapped close to major genes or quantitative trait loci for resistance to scab and mildew previously identified in different apple progenies. Several markers could become efficient tools for marker-assisted selection once converted into breeder-friendly markers. This study demonstrates the efficiency of the NBS-profiling method for generating RGA markers for resistance loci in apple.     

A novel wheat alpha-amylase gene (alpha-Amy3)

Summary A genomic clone of a wheat -amylase gene (Amy3/33) was identified, on the basis of hybridisation properties, as different from -Amy1 and -Amy2 genes which had been characterised previously. The nucleotide sequence revealed that this gene has the normal sequence motifs of an active gene and an open reading frame interrupted by two introns. The protein sequence encoded by this open reading frame is recognisably similar to that of -amylase from the -Amy1 and -Amy2 genes and there is high sequence homology in all three proteins at the putative active sites and Ca⁺⁺ binding region. In addition, the introns are at positions equivalent to the position of introns in the -Amy1 and -Amy2 genes. However, the sequence was less similar to -Amy1 and -Amy2 than these are to each other. Southern blot analysis showed that the Amy3/33 DNA is one of a small multigene family carried on a different chromosome (group 5) from either the -Amy1 or -Amy2 genes. A further difference from the -Amy1 and -Amy2 genes was the pattern of expression. Amy3/33 was expressed only in immature grains and, unlike the -Amy1 and -Amy2 genes, not at all in germinating aleurones. These data suggested therefore that this gene represents a third type of -amylase gene, not described before, which shares a common evolutionary ancestor with the -Amy1 and -Amy2 genes.