Fig. 4. Molecular characteristics of the Arabidopsis RPS5 gene family. (A) Alignment of the extreme C terminus of RPS5 proteins from different species shows a strong conservation of primary structure. AtRPS5A, GenBank ID N37913; AtRPS5B, GenBank ID H35978; CaRPS5, Cicer arietinum (GenBank ID AJ005346); ScRPS5, Saccharomyces cerevisiae (GenBank ID X89368); CeRPS5, Caenorhabditis elegans (GenBank ID P49041); DmRPS5, Drosophila melanogaster (GenBank ID U48394); and HsRPS5, Homo sapiens (GenBank ID MN001009). The fusion of RPS5A and GUS in line 826 (aml1) is shown in the bottom row with the connecting border sequence in italics and the GUS sequence in bold. The number between brackets indicates the position in the protein relative to the N terminus. The asterisks indicate plant-specific residues. (B) Gene structure of AtRPS5A and the aml1 allele. The AtRPS5A gene contains five exons, interrupted by four introns. Arrows I, II and III indicate the positions of PCR-primers. (C) Complementation of the aml1 mutant with a wild-type RPS5A gene copy. The top panel shows the result of a duplex-PCR to detect the wild-type (1.3 kb) and aml1 (0.8 kb) alleles of the RPS5A gene in seedlings. From left to right: C24 wild-type plants, the aml1 mutant and ten independent primary transformants with the MOG-RPS5A construct. Plant 1 is homozygous for the aml1 allele. Bottom panel shows the presence of the NPTII gene located on the MOG-RPS5A construct. (D) Intron and exon sizes (numbering is in the 5' to 3' direction of the genes) are extremely conserved between AtRPS5A and AtRPS5B. (E) RT-PCR analysis using AtRPS5 and AtRPS5B gene-specific primer sets. AtRPS5A primers (left panel) amplify a 0.4 kb fragment from seedling RNA (R) to higher levels than do AtRPS5B specific primers (R, right panel). Control PCR reactions using chromosomal DNA (C) indicate equal efficiency of both primer pairs.
