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Identification of the alternative splicing of the UL49 locus of human cytomegalovirus.

Yang G, Li W, Liao W, Zhang X, Zou Y, Dai J, Li Y, Jing C, Zhou T - Biomed Res Int (2015)

Bottom Line: All the new EST sequences and UL49Y cDNA sequence have been deposited in the GenBank database (GenBank Accession nos.GW314860-GW314900 and GU376796).This study provides us with very important clues for revealing the importance of the UL49 locus alternative splicing.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, College of Life Science and Technology, Jinan University, Guangzhou 510632, China ; Department of Parasitology, Medical School, Jinan University, Guangzhou 510632, China.

ABSTRACT
The UL49 ORF of human cytomegalovirus (HCMV) is essential for viral replication; conserved among all herpes viruses; however, the function is unclear. Once the UL49 ORF was precisely deleted from the start to stop codon, the mutant did not yield infectious progeny. In this study, we find out many alternatively processed ESTs in UL49 locus in HCMV-infected cells, in which there are two novel transcription termination sites in UL49 locus. Most of these ESTs are rare transcripts that contain directed repeat sequences in the intron splicing regions. There is a typical GU-AG intron splicing site in UL49Y transcripts. The 1847 bp UL49Y cDNA spans an ORF from 335 to 1618 and encodes a putative protein of 427 amino acids with a predicted molecular mass of 47.1 kDa. All the new EST sequences and UL49Y cDNA sequence have been deposited in the GenBank database (GenBank Accession nos. GW314860-GW314900 and GU376796). This study provides us with very important clues for revealing the importance of the UL49 locus alternative splicing.

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Organization and transcription summary of HCMV Towne UL49 locus. (a) Nucleotide positions correspond to the genome sequence under GenBank Accession no. AY315197.2. (b) ORF map summary of the UL48, UL48A, UL49, and UL50. (c) cDNAs and the location of polyadenylation signals and polyadenylation site are identified in group B. (d) cDNAs and the location of polyadenylation signals polyadenylation site are identified in group A. (e) Primers (Table 1) corresponding to the nucleotide position of HCMV Towne genome used in this paper.
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fig1: Organization and transcription summary of HCMV Towne UL49 locus. (a) Nucleotide positions correspond to the genome sequence under GenBank Accession no. AY315197.2. (b) ORF map summary of the UL48, UL48A, UL49, and UL50. (c) cDNAs and the location of polyadenylation signals and polyadenylation site are identified in group B. (d) cDNAs and the location of polyadenylation signals polyadenylation site are identified in group A. (e) Primers (Table 1) corresponding to the nucleotide position of HCMV Towne genome used in this paper.

Mentions: To define the 3′ end of the UL49 locus alternative mRNAs, we carried out the RACE method with 3′-Full RACE Core Sets (Takara) and all the primers have been listed in Table 1 and Figure 1. The cDNA template was synthesized with the Oligo dT-3sites Adaptor Primer of the 3′-Full RACE Core Set according to the manufacturer's instructions. The 1st PCR was performed with the 3′ RACE Outer primer (5′-TAC CGT CGT TCC ACT AGT GAT TT-3′) and Primer 1 in 50 μL of the following reaction: 1 × LA PCR Buffer, 0.4 mM dNTP mixture, 0.2 μM each primer, 2.5 U LA Taq polymerase, and 1 μL of cDNA template. PCR amplification was done at 95°C for 5 min and followed by 30 cycles at 94°C for 30 s, 55°C for 1 min, and 72°C for 3 min. The 2nd PCR reactions and conditions were the same as the 1st PCR except for using the 3′RACE Inner primer (5′-CGC GGA TCC TCC ACT AGT GAT TTC ACT ATA GG-3′) and Primer 2, 1 μL the outer PCR product as the template. Each sample was analyzed on a 1.5% agarose gel. Gel-purify of the PCR products were performed by using Gel Extraction Kit (E.Z.N.A.). Then all the isolated fragments were directly cloned into the TA-vector pMD18-T (TaKaRa) and sequenced using the sequencing Primer RV-M and Primer M13-47. Through the 3′-RACE PCR approaches, 10 novel cDNA fragments were cloned, with length from 188 bp to 1594 bp. These new EST sequences have been deposited in the GenBank database (GenBank Accession no. GW314860-GW314869) (Table 2). The various sequences comprised two groups. The group was characterized by the usage of a distinct alternative poly(A) sites within the 3′ untranslated region (3′UTR). Group A has two polyadenylation signals (AATAAA) located at 68947-68942 and 68648-68643 with the polyadenylation site at 68629. Group B has one polyadenylation signal (AATAAA) located at 69643-69638 with the polyadenylation site at 69610 (Figure 1).


Identification of the alternative splicing of the UL49 locus of human cytomegalovirus.

Yang G, Li W, Liao W, Zhang X, Zou Y, Dai J, Li Y, Jing C, Zhou T - Biomed Res Int (2015)

Organization and transcription summary of HCMV Towne UL49 locus. (a) Nucleotide positions correspond to the genome sequence under GenBank Accession no. AY315197.2. (b) ORF map summary of the UL48, UL48A, UL49, and UL50. (c) cDNAs and the location of polyadenylation signals and polyadenylation site are identified in group B. (d) cDNAs and the location of polyadenylation signals polyadenylation site are identified in group A. (e) Primers (Table 1) corresponding to the nucleotide position of HCMV Towne genome used in this paper.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4383306&req=5

fig1: Organization and transcription summary of HCMV Towne UL49 locus. (a) Nucleotide positions correspond to the genome sequence under GenBank Accession no. AY315197.2. (b) ORF map summary of the UL48, UL48A, UL49, and UL50. (c) cDNAs and the location of polyadenylation signals and polyadenylation site are identified in group B. (d) cDNAs and the location of polyadenylation signals polyadenylation site are identified in group A. (e) Primers (Table 1) corresponding to the nucleotide position of HCMV Towne genome used in this paper.
Mentions: To define the 3′ end of the UL49 locus alternative mRNAs, we carried out the RACE method with 3′-Full RACE Core Sets (Takara) and all the primers have been listed in Table 1 and Figure 1. The cDNA template was synthesized with the Oligo dT-3sites Adaptor Primer of the 3′-Full RACE Core Set according to the manufacturer's instructions. The 1st PCR was performed with the 3′ RACE Outer primer (5′-TAC CGT CGT TCC ACT AGT GAT TT-3′) and Primer 1 in 50 μL of the following reaction: 1 × LA PCR Buffer, 0.4 mM dNTP mixture, 0.2 μM each primer, 2.5 U LA Taq polymerase, and 1 μL of cDNA template. PCR amplification was done at 95°C for 5 min and followed by 30 cycles at 94°C for 30 s, 55°C for 1 min, and 72°C for 3 min. The 2nd PCR reactions and conditions were the same as the 1st PCR except for using the 3′RACE Inner primer (5′-CGC GGA TCC TCC ACT AGT GAT TTC ACT ATA GG-3′) and Primer 2, 1 μL the outer PCR product as the template. Each sample was analyzed on a 1.5% agarose gel. Gel-purify of the PCR products were performed by using Gel Extraction Kit (E.Z.N.A.). Then all the isolated fragments were directly cloned into the TA-vector pMD18-T (TaKaRa) and sequenced using the sequencing Primer RV-M and Primer M13-47. Through the 3′-RACE PCR approaches, 10 novel cDNA fragments were cloned, with length from 188 bp to 1594 bp. These new EST sequences have been deposited in the GenBank database (GenBank Accession no. GW314860-GW314869) (Table 2). The various sequences comprised two groups. The group was characterized by the usage of a distinct alternative poly(A) sites within the 3′ untranslated region (3′UTR). Group A has two polyadenylation signals (AATAAA) located at 68947-68942 and 68648-68643 with the polyadenylation site at 68629. Group B has one polyadenylation signal (AATAAA) located at 69643-69638 with the polyadenylation site at 69610 (Figure 1).

Bottom Line: All the new EST sequences and UL49Y cDNA sequence have been deposited in the GenBank database (GenBank Accession nos.GW314860-GW314900 and GU376796).This study provides us with very important clues for revealing the importance of the UL49 locus alternative splicing.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, College of Life Science and Technology, Jinan University, Guangzhou 510632, China ; Department of Parasitology, Medical School, Jinan University, Guangzhou 510632, China.

ABSTRACT
The UL49 ORF of human cytomegalovirus (HCMV) is essential for viral replication; conserved among all herpes viruses; however, the function is unclear. Once the UL49 ORF was precisely deleted from the start to stop codon, the mutant did not yield infectious progeny. In this study, we find out many alternatively processed ESTs in UL49 locus in HCMV-infected cells, in which there are two novel transcription termination sites in UL49 locus. Most of these ESTs are rare transcripts that contain directed repeat sequences in the intron splicing regions. There is a typical GU-AG intron splicing site in UL49Y transcripts. The 1847 bp UL49Y cDNA spans an ORF from 335 to 1618 and encodes a putative protein of 427 amino acids with a predicted molecular mass of 47.1 kDa. All the new EST sequences and UL49Y cDNA sequence have been deposited in the GenBank database (GenBank Accession nos. GW314860-GW314900 and GU376796). This study provides us with very important clues for revealing the importance of the UL49 locus alternative splicing.

Show MeSH
Related in: MedlinePlus