Bioinformatic analyses of GRAS genes in Betula kirghisorum based on transcriptome data.

The transcriptomes of salt-stressed and unstressed Betula kirghisorum plants were analyzed using high throughput sequencing technology. A total of 52,239,804 and 51,772,998 clean reads were obtained from the two libraries, respectively, and de novo assembled into 60,545 all-unigenes. A total of 39,997 unigenes were annotated using public databases. Overall, 7206 genes were differentially expressed in unigenes and were involved in 127 pathways. Thirteen transcription factor families were identified in B. kirghisorum, including GRAS proteins, which are plant-specific transcription factors. By using bioinformatic methods to predict and analyze physicochemical properties, structural data were obtained on the 19 potential GRAS proteins. The results revealed that these proteins are hydrophilic, with significant differences in their length and molecular weight. The main secondary structures were alpha helices and random coils. BkGRAS proteins possess typical GRAS domains: LHR I; VHIID motif; LHR II; PFYRE motif; and SAW motif. In the majority of BkGRAS proteins, AGG, AGA, UCU, GCU, GGG, CCA, GUU, GUG, AUU, GAU, and AAG codons were used preferentially. Aside from the BkGRAS17 gene (relative synonymous codon usage (RSCU) = 1.20), usage of the UUA codon by other BkGRAS genes was low (RSCU < 1.0). The effective number of codons showed that BkGRAS genes have low codon bias. Subcellular localization analysis that predicted these proteins are found in the nucleus, cytoplasm, or chloroplast. BkGRAS proteins were divided into six subfamilies: SCR, LISCL, SCL3, DELLA, HAM, and PAT1. These results provide important information for the further functional study of GRAS genes in B. kirghisorum.

Cloning and expression pattern analysis of BkGRAS2 from Betula kirghisorum.

GRAS proteins are plant-specific transcription factors that are involved in the regulation of root and shoot growth. Here, we cloned BkGRAS2 from Betula kirghisorum (abbreviated to Bk) and analyzed the physicochemical properties and expression pattern of the encoded protein. BkGRAS2 had an open reading frame of 1614 bp encoding 537 amino acid residues. The deduced BkGRAS2 protein was hydrophilic, and it contained highly conserved VHIID and SAW motifs. BkGRAS1 and BkGRAS2 showed considerable sequence similarities. An expression analysis indicated that BkGRAS2 was expressed in root, stem, and leaf, with the highest level in the leaf. Expression of BkGRAS2 was increased following stress treatment with 0.6% NaHCO3. Transient expression analysis of GFP-BkGRAS2 in onion epidermal cells revealed that the BkGRAS2 protein was localized in the cytoplasm, but could also be detected in the nucleus. Our study provides the basis for future research on the role of the GRAS gene family in B. kirghisorum.

Genetic variations of EBV-LMP1 from nasopharyngeal carcinoma biopsies: potential loss of T cell epitopes.

To find Epstein-Barr virus (EBV) strains with genetic variations of EBV latent membrane protein 1 (EBV-LMP1) from nasopharyngeal carcinoma (NPC), the full-length DNA of LMP1 genes from 21 NPC biopsies obtained in Hunan province in southern China was amplified and sequenced. Our sequences were compared to those previously reported by the Clustal V method. Results showed that all 21 sequences displayed two amino acid changes most frequently in LMP1 of CD4+ T cell epitopes at codons 144 (F-->I, 21/21) and 212 (G-->S, 19/21) or (G-->N, 2/21). We also show that type A EBV strain is prevalent in the cases of NPC from Hunan province with a 30-bp 18/21 deletion, and we highlight that this deletion resulted in loss of one of the CD4+ T cell-restricted epitopes. The other 3 sequences without this deletion all had a change at codon 344 (G-->D). Furthermore, in the major epitope sequence of CD8+ T cells restricted by HLA-A2, all 21 sequences showed changes at codons 126 (L-->F) and 129 (M-->I). Our study discovered that one of the 21 sequence variations harbored a new change at codon 131 (W-->C), and 5/21 specimens showed another novel change at codon 115 (G-->A) in the major epitope sequence of CD8+ T cells restricted by HLA-A2. Our study suggests that these sequence variations of NPC-derived LMP1 may lead to a potential escape from host cell immune recognition, protecting latent EBV infection and causing an increase in tumorigenicity.

Genetic variations of EBV-LMP1 from nasopharyngeal carcinoma biopsies: potential loss of T cell epitopes

To find Epstein-Barr virus (EBV) strains with genetic variations of EBV latent membrane protein 1 (EBV-LMP1) from nasopharyngeal carcinoma (NPC), the full-length DNA of LMP1 genes from 21 NPC biopsies obtained in Hunan province in southern China was amplified and sequenced. Our sequences were compared to those previously reported by the Clustal V method. Results showed that all 21 sequences displayed two amino acid changes most frequently in LMP1 of CD4+ T cell epitopes at codons 144 (F arrow right I, 21/21) and 212 (G arrow right S, 19/21) or (G arrow right N, 2/21). We also show that type A EBV strain is prevalent in the cases of NPC from Hunan province with a 30-bp 18/21 deletion, and we highlight that this deletion resulted in loss of one of the CD4+ T cell-restricted epitopes. The other 3 sequences without this deletion all had a change at codon 344 (G arrow right D). Furthermore, in the major epitope sequence of CD8+ T cells restricted by HLA-A2, all 21 sequences showed changes at codons 126 (L arrow right F) and 129 (M arrow right I). Our study discovered that one of the 21 sequence variations harbored a new change at codon 131 (W arrow right C), and 5/21 specimens showed another novel change at codon 115 (G arrow right A) in the major epitope sequence of CD8+ T cells restricted by HLA-A2. Our study suggests that these sequence variations of NPC-derived LMP1 may lead to a potential escape from host cell immune recognition, protecting latent EBV infection and causing an increase in tumorigenicity.