A novel tri-primer polymerase chain reaction method (TP-PCR) was developed for the construction of a fused fpg gene, in which no endonuclease and ligase were used. Instead, two templates and three specifically designed primers were applied. Results showed that pheB and gfp genes, which encodes the catechol 2,3-dioxygenase and the green fluorescent protein (GFP), respectively, were successfully fused into an fpg gene through the rapid TP-PCR system, indicating that TP-PCR method could be a useful tool for DNA fragment fusion in which no proper endonuclease sites were available.

A comparison of Arabidopsis DNA sequences revealed that the final nucleotides at the 3 end of approximately half of the Arabidopsis mRNAs, immediately upstream of the poly(A) tail, differ from the corresponding genomic sequences. This suggests that extra nucleotides were added to these mRNAs at their 3 termini prior to polyadenylation. Among the mRNAs containing additional nucleotides, approximately 65% had a single additional nucleotide, with the nucleotide C added most often. This nontemplated addition before the addition of the poly(A) sequence could be a major contributing factor to the often observed heterogeneity in transcription products. These findings should be helpful in the elucidation of the mechanisms of mRNA 3-end processing.

A novel pollen-specific full-length cDNA clone PSG076 was isolated using suppression subtractive hybridization and 5'/3' RACE techniques. PSG076 was shown to exhibit multi-site polyadenylation by sequencing the 3' ends of the cDNAs. At least six transcripts with different length were produced from the single gene based on different poly(A) tail attachment sites. However, polyadenylation consensus sequence AAUAAA was not seen at the 3'-untranslated sequence. PSG076 contained a 299 bp 5' untranslated region and an open reading frame of 663 bp encoding a 221 amino acid peptide with pI of 4.31. A blast search revealed that this sequence did not show a significant similarity to any genes deposited in the public database. Southern blot indicated that PSG076 was a single copy gene. Northern blot and RT-PCR analysis indicated that PSG076 transcripts showed specific expression in mature pollen, and weak or undetectable signals in uninucleate microspore, immature seed, stem, young leave, root and ovary. Further analysis of the expression pattern in gametophyte showed that PSG076 transcripts were undetectable in uninucleate, binucleate microspore and pollen at early stage, and were first detectable and increased rapidly at middle and late stages of pollen development with the maximum level in mature pollen and also expressed in germinating pollen in vivo, suggesting that PSG076 might play a role in pollen germination and pollen tube growth in addition to its function in maturation. The evidences gathered in this work indicated that the six different transcripts from the single gene were differentially expressed during pollen development.

Here we report the isolation and expression of a pollen-specific cDNA TaPSG719 with an unusually long 5' leader sequence via suppression subtractive hybridization and 5'/3' RACE techniques. The insert in the TaPSG719 is 1 172-bp long, and encodes a protein of 188 amino acids long with a pI of 12.1. This sequence did not show a significant homology to any genes deposited in the public database by BLAST search. Southern blot indicated that TaPSG719 might be multicopy. Northern blot and RT-PCR analyses indicated that TaPSG719 transcripts were specific for mature pollen, and undetectable in microspore, immature seed, stem, young leaf, root and ovary. During pollen development, TaPSG719 transcripts were first detectable on the 5th day before anthesis and increased rapidly at middle stages of pollen development with maximum levels on the 4th day before anthesis, and decreased during pollen maturation. It is noted that TaPSG719 contains an unusually long 5' leader sequence (329-nt) upstream the ATG start codon, suggesting that the gene could be subject to translational regulation. To investigate the role of the 5' UTR on translation, in vitro transcription/translation assays with various deletion and mutation constructs were performed using wheat germ extract. The results demonstrated that the 5' UTR affected positively downstream translation in wheat germ extract.