Department of Biochemistry, Medical University of South Carolina, 141 Ashley Avenue, Charleston,
South Carolina 29425, USA
*Present address: School of Life Sciences, Jawaharlal Nehru University, New Delhi 110 067, India

(Fax, 91-11-6866886; Email, pradip12@hotmail.com)

Accumulation of immunoglobulin Ig RNA (from several loci viz., C_H, Ca , J_k–C_k and Sm during Igm isotype switching) in B cells and T cell receptor (TCR) RNAs (a , b and g  ) in T cells of unusual sizes emanating from germline and rearranged genes were reported to accumulate in human and mouse (and murine too). The precise mechanism and function of these sterile RNA species are yet to be delineated. Similar accumulation of RNA species of unusual sizes were identified with DNA–RNA hybridization and isolation of cDNA employing with DNA and antibody probes in mouse hybridoma, murine tumour, non-human primate marmoset tumour and human leukemic cells.

1. Introduction

Accumulation of Ig RNA (from several loci viz., C_H, Ca , J_k–C_k and Sm during Igm isotype switching) in B cells and TCR RNAs (a , b and g  ) in T cells of unusual sizes emanating from germline and rearranged genes were reported in cell lines, cell clones, suppressor hybridomas and tissues of human, mouse and murine origins including transgenic constructs in mouse tissues (Fondell and Marcu 1992; Goldman et al 1993; Qian et al 1993a, b; Okade et al 1994; Soloff et al 1995; Ktorza et al 1996; Sleckman et al 1996; Villey et al 1997; Linehan et al 1998; Müller et al 1998; Sikes et al 1998). No precise function has been ascribed to this RNA accumulation to date. Based on an analysis with DNA sequencing RT–PCR amplified product with sequence-specific oligonucleotides and DNA–RNA hybridization with exon/intron sequences (separately or in combination), these TCRb mRNAs were classified in four status: (i) unspliced pre-mRNAs (6–5 kb VDJC), (ii) intermediate RNAs (ranging from 4 kb to 1·4 kb), (iii) spliced mature mRNAs (1·3 kb VDJC or VJC, 1·1 kb DJC, 1·0 kb DJC or JC, 1·0 kb unrearranged DJ) as well as (iv) spliced lower mRNA (ranging from 0·9 kb to 0·18 kb) which mostly contain C exons in association with one or more C introns. The presence of four introns (IVS, IVS1 and IVS2 and IVS3) and usually long polyA-tail (characteristic of nuclear transcript) were found in the unrearranged RNA species of four groups. The Cb introns, IVS1Cb ₁ and IVS2Cb ₂ are excised as stable lariat molecules that accumulate in the nucleus of T cells (Qian et al 1993a, b). Both germline and rearranged Vb ₁₄ transcripts were also reported from expression of Vb  Jb  Cm transgene construct containing enhancer (Em or Eb  ) in tissues of mouse (Okade et al 1994). The different promoters, enhancers, transcription-specific regulators (cis or trans), Igm isotype switching involving RNA at Sm (in B cells), splicing status in T-cells and correlation of transcript accumulation with V(D)JC rearrangement (or without rearrangement in some T cells) involving a common recombinase in precursor (pre-) stages of both B and T cells were discussed in detail (Yoshikai et al 1984; Kronenberg et al 1985; Qian et al 1993a, b; Müller et al 1998). In this study, sterile TCBb -related RNA were identified by DNA–RNA hybridization and selection of cDNA with DNA and antibody probes, in hybridoma, tumour and leukemic cells of human, rodent and primate origins.

2. Materials and methods

2.1 Cell culture and other methods

The azobenzenearsonate hapten-specific mouse hybridoma 51H7D, BALB/C-derived monoclonal murine tumour hybrid clone WEHI-7, non-human primate marmoset (Sanguinus oedipus) tumour 70N2 and human leukemic MOLT-3 cells were described previously (Yoshikai et al 1984; Kronenberg et al 1985; Schluter and Marchalonis 1986; Bristow and Marchalonis 1989; Hubbard et al 1989; Cone and Marchalonis 1993; Bhattacharya 1998). The conditions for cell culture (Schluter and Marchalonis 1986), isolation of polyA and total RNA after guanidium isothyacyanate/CsCl density gradient centrifugation following oligo-dt cellulose chromatography, DNA–RNA hybridization, cDNA library in l gt11, screening of cDNA with both DNA and antibody, plasmid purification by CsCl/ethidium bromide density gradient centrifugation, electroelution of restriction enzyme fragments from agarose gels, radioisotopes for hybridization and colour substrates for antibody screening were described before (Sambrook et al 1989; Bhattacharya 1998).

2.2 Description of probes

The DNA probes, 770 bp rearranged human jurkat TCRb exon (100 nt to 870 nt) corresponding to Db _1·1Jb _1·1Cb _1·1 and 400 bp (RsaI/HincII) entire rearranged V_kJ_k ligated exon (first 108 amino acids of variable region) of k -chain of phosphocoline-specific human S107 myolema tumour cell and the rabbit affinity-purified antibody directed against Jb peptide sequence (ANYGYTFGSGTRLTVV) of jurkat TCRb exon were described (Kawan et al 1981; Yoshikai et al 1984; Schluter and Marchalonis 1986). The DNA sequence data of jurkat TCRb DJC (b ₁), Vk Jk S107, MOLT-3 TCRb , mouse TCRb and murine TCRb revealed maximum identities in J region (50% to 80% in nt) and C region (80% to 90% in nt), and weak identities (20% to 30% in nt) in V region (although among identities subfamilies of the same species are 20% to 80% in nt) (Kawan et al 1981; Malissen et al 1984; Siu et al 1984; Yoshikai et al 1984; Barth et al 1985; Governan et al 1985; Kronenberg et al 1985; Chou et al 1987; Marchalonis et al 1988). In fact, jurkat TCRb has extensive homology with human l which is expressed in B cells (not in T cells). The identities (in nt) between Vk Jk S107 and jurkat (D)JC were about 75% in J region and weak in other regions. The alignment of DNA sequence is not shown.

3. Results

To determine whether RNA was transcribed from the TCRb locus, total and polyA RNA were isolated from experimental 51H7D and WEHI-7 cells, fractionated on a denaturing gel, blotted to nitrocellulose and hybridized with the DNA probes. Control RNA, isolated from 70N2 and MOLT-3, were electrophoresed in parallel and hybridized similarly. A complex array of RNA accumulation in both polyA and total RNA content of these cells are shown in figure 1. The level of detection was confirmed by high-stringent washing with SDS (1%, 0·5%, 0·1%, 0·05%, 0·01%)-containing buffer several times at 65°C for 15–30 min. This strongly suggests that transcripts of various sizes, which accumulated, were comparatively long lived. If these were short-lived, these would not be detected. In 51H7D and WEHI-7, RNA components (6–2·6 kb) which hybridized with (D)JC (figure 1A), also hybridized with VJ (figure 1B) very weakly because of common J sequence, and these are poly-adenylated. Based on hybridization pattern with DJC(b ) exon and VJ(k ) exon DNA probes. RNA status has been implicated (figure 1) in the order as explained by Qian et al (1993a, b). In murine tumour WEHI-7, RNA species correspond to 6–5 kb unspliced pre-mRNA (VDJC, DJC), 3 kb intermediate, 1·23 kb mature, 1·1 kb (DJC) and 1·0 kb (DJC or JC) RNA. In mouse hybridoma 51H7D, RNA species correspond to 2·6 kb intermediate, 1·23 kb mature, 1·1 kb (DJC) and 1·0 kb (DJC or JC) RNA. In primate marmoset tumour 70N2, RNA species correspond to 1·4 kb intermediate, 1·23 kb intermediate, 1·1 kb (DJC), 1·0 kb (DJC or JC) and 0·9 kb (C) RNA. In human leukemic MOLT-3 RNA species correspond to 1·23 kb intermediate, 1·1 kb (DJC), 1·0 kb (DJC or JC) and 0·9 kb (C) RNA DJC(b ) DNA exon probe did not identify lower than 0·9 kb RNA containing mostly C introns or explain the existence of intron within complex set of RNA.

As compared to the isolation of genes cDNA from cDNA expression libraries of 51H7D, WEHI-7 and 70N2 (data not presented), exceptionally few cDNA were identified with the rabbit antibody directed against Jb (ANYGYTFGSTRLTVV) peptide. Alternatively, translation was possibly blocked by the intervening untraslated sequence, implying the unrearranged status of most cDNA and the corresponding RNA. DNA sequencing would confirm whether any RNA was transcribed from the rearranged or unrearranged genes. This antibody cannot identify any other b -galactosidase fusion product that excludes J sequence.

4. Discussion

Although the mouse hybridoma 51H7D and murine tumour WEHI-7 cells do not synthesize TCRb chains (Schluter and Marchalonis 1986; Marchalonis et al 1988; Bristow and Marchalonis 1989; Hubbard et al 1989; Cone and Marchalonis 1993), they do accumulate TCRb RNA like marmoset 70N2 and human MOLT-3, both of which synthesize TCRb chain. This accumulation of RNA displays a mock situation that may not be an accidental artefact or may have a definite function in the precursor (pre)-T cell stage. More precisely, the complex array of RNA species mimic an event caused by RNA processing where spliced and unspliced products accumulate in similar fashion (Qian et al 1993a, b). The RNA purification by guanidium isothyacyanate did enrich RNA but eliminated the barrier between nuclei and cytoplasm. This revelation is only justified to assess activation of transcription rather than to distinguish the specific localization of RNA species in the two compartments.

Acknowledgements

The author wishes to thank Dr John J Marchalonis for providing post-doctoral fellowship from Grant CA-42049 supported by National Institute of Health, Dr Leo Chao for advice, Dr Greg Warr for Vk Jk S107 DNA and Dr Tak W Mak (Department of Biophysics, University of Toronto, Canada) for DJC(b ) DNA.

MS received 15 January 1999; accepted 3 August 1999