Introduction of a plant intron into the luciferase gene ofPhotinus pyralis

We describe a new luciferase reporter gene,luc ^INT, for early detection of luciferase activity inAgrobacterium transformation studies, and present improved techniques for the extraction of luciferase that decrease the time needed to quantitate luciferase activity. Theluc ^INT reporter gene combines the PIV2 intron fromGUS ^INT withluc ^*, the modified luciferase gene.luc ^INTis expressed in plant cells but not inAgrobacterium, allowing earlier detection of gene expression in the presence ofAgrobacterium during transformations in tobacco leaf discs. Stable expression levels ofluc ^INT andluc ^* in tobacco suspension cultures are compared for two different promoters. The nucleotide sequence data for the gene will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number U84006.     

Construction and testing of an intron-containing luciferase reporter gene from<Emphasis Type="Italic">Renilla reniformis</Emphasis>

We describe a newRenilla reniformis luciferase reporter gene,RiLUC, which was designed to allow detection of luciferase activity in studies involvingAgrobacterium-based transient expression studies. TheRLUC gene was altered to contain a modified intron from the castor bean catalase gene while maintaining consensus eukaryotic splicing sites recognized by the plant spliceosome.RLUC andRiLUC reporter genes were fused to the synthetic plant SUPER promoter. Luciferase activity within agrobacteria containing the SUPER-RLUC construct increased during growth in culture. In contrast, agrobacteria harboring the SUPER-RiLUC gene fusion showed no detectable luciferase activity. Agrobacteria containing these gene fusions were cotransformed with a compatible normalization plasmid containing a cauliflower mosaic virus 35S promoter (CaMV) joined to the firefly luciferase coding region (FiLUC) and infused into tobacco leaf tissues through stomatal openings. The kinetics of luciferase production from theRLUC orRiLUC reporters were consistent, with expression of theRiLUC gene being limited to transiently transformed plant cells.RiLUC activity from the reporter gene fusions was measured transiently and within stably transformed tobacco leaf tissues. Analysis of stably transformed tobacco plants harboring either reporter gene fusion showed that the intron altered neither the levels of luciferase activity nor tissue-specific expression patterns driven by the SUPER promoter. These results demonstrate that theRiLUC reporter gene can be used to monitor luciferase expression in transient and stable transformation experiments without interference from contaminating agrobacteria.     

Investigation of the RH locus in gorillas and chimpanzees

The human Rh blood-group system is encoded by two homologous genes,RhD andRhCE. TheRH genes in gorillas and chimpanzees were investigated to delineate the phylogeny of the humanRH genes. Southern blot analysis with an exon 7-specific probe suggested that gorillas have more than twoRH genes, as has recently been reported for chimpanzees. Exon 7 was well conserved between humans, gorillas, and chimpanzees, although the exon 7 nucleotide sequences from gorillas were more similar to the humanD gene, whereas the nucleotide sequences of this exon in chimpanzees were more similar to the humanCE gene. The intron between exon 4 and exon 5 is polymorphic and can be used to distinguish the humanD gene from theCE gene. Nucleotide sequencing revealed that the basis for the intron polymorphism is anAlu element inCE which is not present in theD gene. Examination of gorilla and chimpanzee genomic DNA for this intron polymorphism demonstrated that theD intron was present in all the chimpanzees and in all but one gorilla. TheCE intron was found in three of six gorillas, but in none of the seven chimpanzees. Sequence data suggested that theAlu element might have previously been present in the chimpanzeeRH genes but was eliminated by excision or recombination. Conservation of theRhD gene was also apparent from the complete identity between the 3′-noncoding region of the human D cDNA and a gorilla genomic clone, including anAlu element which is present in both species. The data suggest that at least twoRH genes were present in a common ancestor of humans, chimpanzees, and gorillas, and that additionalRH gene duplication has taken place in gorillas and chimpanzees. TheRhCE gene appears to have diverged more thanRhD among primates. In addition, theRhD gene deletion associated with the Rh-negative phenotype in humans seems to have occurred after speciation. Correspondence to: C.M. Westhoff     

Red cell H-deficient,salivary ABH secretor phenotype of Reunion island. Genetic control of the expression of H antigen in the skin

Based on the genetic model proposing thatH andSe are two structural genes, we predicted that the red cell H-deficient, salivary ABH secretor phenotype should be found on Reunion island, where a large series of H-deficient non-secretor families have been previously described. Two such Reunion individuals are now reported. POU [A_h, Le(a–b+), secretor of A, H, Le^a and Le^b in saliva] and SOU [O_h, Le(a–b+), secretor of H, Le^a and Le^b in saliva]. Both are devoid of H -2-fucosyltransferase activity in serum. In addition, the preparation of total non-acid glycosphingolipids from plasma and red cells of POU revealed the type 1ALe^b heptaglycosylceramide and small amounts of the monofucosylated type 1 A hexaglycosylceramide. Both glycolipids possess an H structure probably synthesised by the product of theSe gene. No other blood group A glycolipids, with types 2, 3 or 4 chains, normally present in the presence of the product of theH gene, were found on red cells or plasma of POU.TheH,Se andLe genetic control of the expression of ABH and related antigens in different tissue structures of the skin is described in 54 H-normal individuals of known ABO, secretor and Lewis phenotypes; in one red cell H-deficient salivary secretor (SOU); and in one H-deficient non-secretor (FRA). Sweat glands express ABH under the control of theSe gene. Sweat ducts express ABH under the control of bothH andSe genes and Lewis antigens under the control ofLe and bothH andSe genes. Epidermis, vascular endothelium and red cells express ABH under the control of theH gene. The products ofH andSe genes are usually expressed in different cells. However, the results illustrate that in some structures, like the epithelial cells of sweat ducts, both the products ofH andSe genes can contribute to the synthesis of the same Le^b structure.     

Carbohydrate uptake genes inEscherichia coli are induced by carbon starvation

Escherichia coli produces several membrane-associated and periplasmic proteins in response to deprivation for a carbon source. The carbon starvation response involves a two- to fourfold, cAMP-dependent induction of operons involved in carbohydrate uptake and utilization, including thelac operon. Threecarbon-starvation-inducible (sci) gene fusions to aphoA reporter sequence were characterized. ThephoA-fusions werecya ⁺/crp ⁺-dependent and located in three previously characterized genes involved in high-affinity uptake of alternative carbon sources:mglB, encoding the periplasmic galactose binding protein;rbsB, encoding the periplasmic ribose binding protein; andlamB, encoding the maltodextrin-specific outer membrane porin.     

Therps16 intron and the phylogeny of theRubioideae (Rubiaceae)

The phylogeny of the subfamilyRubioideae (Rubiaceae) was estimated from sequence variation in therps16 intron (cpDNA) in 143 ingroup and 5 outgroup taxa. The analysis largely confirms a recent one based onrbcL sequences, but branch support is often much stronger. Three of the traditional subfamilies are supported,Rubioideae, Cinchonoideae s. str., andIxoroideae s. l. while there is no support forAntirheoideae. TheRubioideae are the sister group of all otherRubiaceae and comprise the tribesAnthospermeae, Coccocypseleae, Cruckshanksieae, Coussareeae, Gaertnereae, Hedyotideae, Knoxieae, Morindeae, Ophiorrhizeae, Paederieae, Pauridiantheae, Perameae, Psychotrieae, Rubieae, Spermacoceae, Theligoneae, andUrophylleae. TheHamelieae andHillieae belong to theCinchonoideae. Rachicallis andSiemensia should be transferred from theHedyotideae to theCinchonoideae. ThePauridiantheae, Urophylleae, Ophiorrhizeae, andRaritebe form the basalmost subclade of theRubioideae. The second basalmost clade consists of the generaLasianthus andPerama. The third basalmost clade consists of the tribesCoussareeae, Coccocypseleae andCruckshanksieae, and the generaDeclieuxia andHindsia. The tribesKnoxieae, Anthospermeae, Argostemmateae, Paederieae, Theligoneae, Rubieae, Hedyotideae, andSpermacoceae are members of one clade. TheKnoxieae are monophyletic ifOtiophora, Otomeria, andPentas are included. The tribeAnthospermeae is supported as monophyletic, but its subtribes are not. ThePaederieae, together withTheligonum, form a paraphyletic grade basal to theRubieae. TheHedyotideae, includingSchismatoclada, form a grade at the base of theSpermacoceae. TheGaertnereae are monophyletic and distinct from thePsychotrieae. TheMorindeae are monophyletic and includeDamnacanthus andMitchella. Schradera is the sister group of theMorindeae. ThePsychotrieae are monophyletic when theGaertnereae, Lasianthus, andDeclieuxia are excluded. The recognition of a subtribeHydnophytineae leaves the rest of thePsychotrieae paraphyletic.Psychotria is paraphyletic with respect to all other genera of the tribe. Approximately 50 genera are here classified for the first time based on molecular data.     

Evolutionary relationship ofHLA-DRB genes inferred from intron sequences

The major histocompatibility complex (Mhc) consists of class I and class II genes. In the humanMhc (HLA) class II genes, nineDRB loci have been identified. To elucidate the origin of these duplicated loci and allelic divergences at the most polymorphicDRBI locus, introns 4 and 5 as well as the 3′ untranslated region (altogether approximately 1,000 base pairs) of sevenHLA-DRB loci, threeHLA-DRBI alleles, and nine nonhuman primateDRB genes were examined. It is shown that there were two major diversification events inHLA-DRB genes, each involving gene duplications and allelic divergences. Approximately 50 million years (my) ago,DRBI ^*04 and an ancestor of theDRB1 ^*03 cluster (DRBI ^*03, DRBI^*15, andDRB3) diverged from each other andDRB5, DRB7, DRB8, and an ancestor of theDRB2 cluster (DRB2, DRB4, andDRB6) arose by gene duplication. Later, about 25 my ago,DRBI ^*15 diverged fromDRBI*03, andDRB3 was duplicated fromDRBI ^*03. Then, some 20 my ago, the lineage leading to theDRB2 cluster produced two new loci,DRB4 andDRB6. TheDRBI ^*03 andDRBI ^*04 allelic lineages are extraordinarily old and have persisted longer than some duplicated genes. The orthologous relationships ofDRB genes between human and Old World monkeys are apparent, but those between Catarrhini and New World monkeys are equivocal because of a rather rapid expansion and contraction of primateDRB genes by duplication and deletion. Correspondence to: Y. Satta     

Evaluation in tobacco of the organ specificity and strength of therolD promoter,domain A of the 35S promoter and the 35S2 promoter

In order to study the expression in plants of therolD promoter ofAgrobacterium rhizogenes, we have constructed chimaeric genes placing the coding region of thegusA (uidA) marker gene under control of tworolD promoter fragments of different length. Similar results were obtained with both genes. Expression studies were carried out in transformed R₁ progeny plants. In mature transformed tobacco plants, therolD-gus genes were expressed strongly in roots, and to much lower levels in stems and leaves. This pattern of expression was transmitted to progeny, though the ratio of the level of expression in roots relative to that in leaves was much lower in young seedlings. The degree of root specificity inrolD-gus transformants was less than that of a gene constructed with domain A of the CaMV 35S promoter,domA-gus, but the level of root expression was much higher than with the latter gene. However, the level of expression of therolD-gus genes was less than that of agus gene with a 35S promoter with doubled domain B, 35S²-gus. TherolD-gus genes had a distinctive pattern of expression in roots, compared to that of the two other genes, with the strongest GUS activity observed in the root elongation zone and in vascular tissue, and much less in the root apex.     

Intron-specific stimulation of anaerobic gene expression and splicing efficiency in maize cells

Most of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes characterized in plants and algae to date have one intron very close to the 5 end of the gene. To study the functional relevance of some of these introns for gene expression we have analysed the influence of three 5 introns on transient gene expression of the anaerobically inducible maizeGapC4 promoter in maize cells. Under aerobic conditions, reporter gene expression is increased in the presence of the first introns of theGapC4 andGapC1 genes, and the first intron of the nuclear encoded chloroplast-specificGapA1 gene. In contrast, theGapC4 intron increases anaerobic gene expression above the level obtained for the intronless construct, while anaerobic expression of constructs harboring theGapA1 andGapC1 introns was similar to the anaerobic expression level of the intronless construct. Splicing analysis revealed that theGapC4 intron is processed more efficiently under anaerobic conditions, while no change in splicing efficiency is observed for theGapC1 and theGapA1 introns when subjected to anaerobic conditions. These results suggest that an increase in splicing efficiency contributes to the anaerobic induction of the maizeGapC4 gene.     

Mutations affecting extracellular protease production in the filamentous fungusAspergillus nidulans

The extracellular proteases ofAspergillus nidulans are known to be regulated by carbon, nitrogen and sulphur metabolite repression. In this study, a mutant with reduced levels of extracellular protease was isolated by screening for loss of halo production on milk plates. Genetic analysis of the mutant showed that it contains a single, recessive mutation, in a gene which we have designatedxprE, located on chromosome VI. ThexprE1 mutation affected the production of extracellular proteases in response to carbon, nitrogen and, to a lesser extent, sulphur limitation. Three reversion mutations,xprF1, xprF2 andxprG1, which suppressxprE1, were characterised. BothxprF andxprG map to chromosome VII but the two genes are unlinked. ThexprF1, xprF2 andxprG1 mutants showed high levels of milk-clearing activity on medium containing milk as a carbon source but reduced growth on a number of nitrogen sources. Evidence is presented that thexprE1 andxprG1 mutations alter expression of more than one protease and affect levels of alkaline protease gene mRNA.     

A molecular genetic linkage map of mouse chromosome 18, includingspm,Grl-1, Fim-2/c-fms,andMbp

Restriction endonuclease fragment length variations (RFLV) were detected in mice with DNA probes for myelin basic protein (Mbp), glucocorticoid receptor-1 (Grl-1), and Friend MuLV integration site-2 (Fim-2). RFLV of theMbp gene were found inSacI restriction patterns, RFLV of theGrl-1 gene were found inEcoRV patterns, and RFLV of theFim-2 were found inBglII patterns. A three-point backcross was carried out by the backcross mating (C57BL/KsJ-spm/spm × MOL-MIT)F₁ males × C57BL/KsJ-spm/spm; spm is an autosomal recessive gene causing sphingomyelinosis. From the results,spm, Grl-1, Fim-2, andMbp loci were mapped on chromosome 18, and the following order of genes is proposed, with distances between genes in parentheses: centromere—spm—(7.8 cM)—Grl-1—(7.8 cM)—Fim-2—(39.1 cM)—Mbp—telomere. All laboratory strains and two European subspecies (Mus mus domesticus andM. m. brevirostris) carry theGrl-1 ^a ,Fim-2 ^a , andMbp ^a alleles. In contrast, another wild subspecies from Europe (M. m. musculus) and some Asian subspecies (M. m. molossinus, Chinese mice of wild origin, andM. m. yamashinai) carry theGrl-1 ^b ,Fim-2 ^b , andMbp ^b alleles. Onlycastaneus strains carry the intermediate combination of theGrl-1 ^b ,Fim-2 ^a , andMbp ^b alleles.     

The effect of spo0 mutations on the expression of spo0A- and spo0F-lacZ fusions

Summary We have constructedspo0A-lacZ andspo0F-lacZ fusions with a temperate phage vector and have investigated howspo0 gene products are involved in the expression of each of these genes. The expression ofspo0A-lacZ andspo0F-lacZ was stimulated at about the time of cessation of vegetative growth in Spo⁺ cells. This stimulation ofspo0A-lacZ was impaired by mutations in thespo0B, D, E, F orH genes but was not affected by mutations in thespo0J orK genes. Similar results were obtained with thespo0F-lacZ fusion. The effect of thespo0A mutation onspo0A-lacZ expression was characteristic: thespo0A-directed β-galactosidase activity found during vegetative growth was significantly enhanced in thespo0A mutant. This result suggests thatspo0A gene expression is autoregulated being repressed by its own gene product. Another remarkable observation was the effect of thesof-1 mutation, which is known to be aspo0A allele; it suppressed the sporulation deficiency ofspo0B, spo0D andspo0F mutants. Thespo0A-lacZ stimulation, which is impaired by any one of thesespo0 mutations, was restored by the additionalsof-1 mutation.     

Expression ofCHS, CHI, andDFR genes in response to light in small radish seedlings

The expression patterns of the genes involved in flavonoid biosynthesis and the changes in anthocyanin content were investigated in small radish (Raphanus sativus L. varsativus) seedlings during light treatment. Anthocyanin content increased until day 4, reaching about 100-fold greater than the control plants, then decreased.CHS (chalcone synthase) mRNA reached a maximum level at 4 h, remained at relatively high levels until day 3, and then decreased rapidly. TheCHI (chalcone isomerase) andDFR (dihydrofolate reductase) mRNA levels reached maximum at 6 h and day 2, respectively, but were decreased rapidly thereafter. All the genes were expressed strongly in hypocotyls, but were either expressed weakly in roots or not expressed at all in cotyledons. Genomic hybridization showed that theCHS gene belonged to a small multigene family, while theCHI andDFR genes were present in one copy per haploid genome.     

Cloning and characterization of theeapB andeapC genes ofCryphonectria parasitica encoding two new acid proteinases, and disruption ofeapC

Two new proteinases secreted byCryphonectria parasitica, namely EapB and EapC, have been purified. The corresponding structural genes were isolated by screening a cosmid library, and sequenced. Comparison of genomic and cDNA sequences revealed that theeapB andeapC genes contain three and two introns, respectively. The products of theeapB andeapC genes as deduced from the nucleotide sequences, are 268 and 269 residues long, respectively. N-terminal amino acid sequencing data indicates that EapC is synthesized as a zymogen, which yields a mature 206-amino acid enzyme after cleavage of the prepro sequence. Similarly, sequence alignment studies suggest that EapB is secreted as a 203-residue form which shares extensive similarities not only with EapC but also with two other acid fungal proteinases. However, they display distinct structural features; for example, no cysteine residue is found in EapC. TheeapC gene was mutated using a two-step gene replacement strategy which allowed the specific introduction of several stop codons at the beginning of theeapC coding sequence in an endothiapepsin-deficient (EapA⁺)C. parasitica strain. Although the resulting strain did not secrete EapC, it still exhibited residual extracellular proteolytic activity, which could be due to EapB.     

A new mutation inEscherichia coli K12,isfA, which is responsible for inhibition of SOS functions

A new mutation inEscherichia coli K12,isfA, is described, which causes inhibition of SOS functions. The mutation, discovered in a ΔpolA ⁺ mutant, is responsible for inhibition of several phenomena related to the SOS response inpolA ⁺ strains: UV- and methyl methanesulfonate-induced mutagenesis, resumption of DNA replication in UV-irradiated cells, cell filamentation, prophage induction and increase in UV sensitivity. TheisfA mutation also significantly reduces UV-induced expression of β-galactosidase fromrecA::lacZ andumuC′::lacZ fusions. The results suggest that theisfA gene product may affect RecA^* coprotease activity and may be involved in the regulation of the termination of the SOS response after completion of DNA repair. TheisfA mutation was localized at 85 min on theE. coli chromosome, and preliminary experiments suggest that it may be dominant to the wild-type allele.