RESUMEN
The complete nucleotide sequence of the intergenic region between the 25 S and 18 S wheat ribosomal RNA genes has been determined from a 4.6 kb EcoRI-BamHI fragment (1 kb = 10(3) bases or base-pairs) subcloned from the plasmid pTa71. Within this subclone the intergenic DNA is flanked by the 3' end of the 25 S and the 5' end of the 18 S ribosomal RNA sequences. Four repeat families are present within the intergenic region. The major repeat family A, consists of 12 direct repeat units of 135 or 136 base-pairs (bp) flanked by diverged truncated copies. Within each A repeat a subrepeat structure has been revealed. Family B, which is localized to the 5' side of the A repeats, contains three repeat units, one of 152 bp, the second of 150 bp and a truncated unit of 107 bp. Family C, which is localized in the transcribed rRNA precursor, consists of two direct repeat units of 172 and 174 bp and possesses some short subrepeat motifs. The C repeats may have evolved by and diverged from one another by the insertion of short transposable sequences. Family D consists of two direct repeat units of 30 bp located 5' to the start of transcription. Statistical analysis of repeat family A showed that there is a significant association between the similarity of any two repeat units and their distance apart in the array. The near identity of members of the A family is maintained presumably by processes such as unequal crossing over and gene conversion, but the members at each end of the array show more divergence. Sequence motifs in the A and C repeat families and in other regions including the 5' end of 18 S RNA are related, implying that much of the intergenic DNA may have evolved from a few short ancestral sequences. The B and D repeats or their equivalent are not found in a maize ribosomal DNA repeat unit. The DNA in the external transcribed spacer DNA 5' to the 18 S RNA sequence is longer in wheat than in maize. This is due principally to two duplications and insertion of a sequence with dyad symmetry in the wheat gene.
Asunto(s)
ADN Ribosómico/genética , Secuencias Repetitivas de Ácidos Nucleicos , Triticum/genética , Secuencia de Bases , Evolución Biológica , Datos de Secuencia Molecular , Regiones Terminadoras Genéticas , Zea mays/genéticaRESUMEN
An earlier report (Baulcombe, D. C., and Buffard, D. (1983) Planta 157, 493-501) described the isolation of cDNA clones from mRNAs which are produced in increased amounts when aleurone layers of wheat are treated with gibberellic acid. It is shown here that for one of those cDNAs (2473) the change in level of mRNA in aleurone parallels the change in level of alpha-amylase mRNA. This result was obtained in experiments where the level of gibberellic acid was varied and also when the mRNA was isolated from wheat genotypes which varied in ability to respond to gibberellic acid. In contrast to this, the pattern of 2437 mRNA accumulation in immature grains and in leaf tissue was quite distinct from the pattern of alpha-amylase mRNA accumulation. Analysis of wheat DNA showed that the 2437 mRNA is encoded by a small family of genes located on the short arm of the group 6 chromosomes. One member of this gene family was cloned and sequenced. The coding sequence is interrupted by eight introns and encodes a protein of Mr 55,433. By using hybridization probes from the 5' exon in an S1 nuclease protection assay it was shown that the 2437 mRNA was produced in aleurones (coordinately with alpha-amylase) and in immature grains (not coordinately with alpha-amylase). However, sequence comparison of 1 kilobase of the 5'-flanking region with the sequence of alpha-amylase genes provided no indication of the regulatory elements which would be active in aleurone cells. The protein sequence deduced from the gene sequence has extensive homology with the yeast carboxypeptidase Y, especially in the active site and substrate binding regions. This homology is greater than with the carboxypeptidase I from barley. It is suggested therefore that there are several types of carboxypeptidase encoded in the cereal genome. The sequence of the 2437 protein would represent one of these types and the barley carboxypeptidase I, another.
Asunto(s)
Carboxipeptidasas/genética , Genes/efectos de los fármacos , Giberelinas/farmacología , Plantas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN/aislamiento & purificación , Enzimas de Restricción del ADN , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , ARN Mensajero/genética , Homología de Secuencia de Ácido Nucleico , Transcripción Genética , Triticum/genéticaRESUMEN
A genomic clone of a wheat alpha-amylase gene (lambdaAmy3/33) was identified, on the basis of hybridisation properties, as different from alpha-Amy1 and alpha-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 alpha-amylase from the alpha-Amy1 and alpha-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 alpha-Amy1 and alpha-Amy2 genes. However, the sequence was less similar to alpha-Amy1 and alpha-Amy2 than these are to each other. Southern blot analysis showed that the lambdaAmy3/33 DNA is one of a small multigene family carried on a different chromosome (group 5) from either the alpha-Amy1 or alpha-Amy2 genes. A further difference from the alpha-Amy1 and alpha-Amy2 genese was the pattern of expression. lambdaAmy3/33 was expreseed only in immature grains and, unlike the alpha-Amy1 and alpha-Amy2 genes, not at all in germinating aleurones. These data suggested therefore that this gene represents a third type of alpha-amylase gene, not described before, which shares a common evolutionary ancestor with the alpha-Amy1 and alpha-Amy2 genes.