Sequence analysis of cloned cDNA encoding part of an immunoglobulin heavy chain

Rogers, J. H.; Clarke, Patrick; Salser, Winston

doi:10.1093/nar/6.10.3305

Cited by 39 publications

(19 citation statements)

References 43 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Alternatively, the splicing pattern of such a transcript may be determined by other factors acting in concert with U-I snRNA alignment of splicing sites. The secondary structure of the hnRNA may sequester some potential splice sites and make others more readily available (42,43). This structure may change as splicing progresses.…”

Section: '-T T T T(t)t T T T T T Ct Tncamentioning

confidence: 99%

A mechanism for RNA splicing.

Rogers¹,

Wall²

1980

Proc. Natl. Acad. Sci. U.S.A.

475

162

View full text Add to dashboard Cite

ABSrRACT The most abundant of the stable small nuclear RNAs of eukaryotic cells, U-1 small nuclear RNA, is exactly complementary to the consensus sequences at RNA splice sites.We propose that this RNA is the recognition component of the nuclear RNA splicing enzyme and forms base pairs with both ends of an intron so as to align them for cutting and splicing.Intramolecular RNA splicing, first discovered for the adenovirus-specific RNA (1-3), occurs in the expression of numerous eukaryotic genes (4). At present there are three known classes of DNA sequences at splice sites, found in genes for a chloroplast tRNA (5), yeast tRNAs (6), and vertebrate mRNAs (7-9). Each class has distinctive sequence characteristics. For the splice sites of vertebrate genes, comparisons of a limited set of sequences (7,8) showed that introns were flanked in every case by 5'-/ G-T-(intron)-A-G/-3'. Subsequent sequences have borne this out (9) and have shown that the 5' ("upstream") and 3' ("downstream") ends of introns have approximately the following consensus or "optimal" sequences: 5'-(exon)-A-G/G-T-A-A-G-T-A-(intron) -T-T-T-T-Y-T-T-T-T-T-T-C-T-T-N-C-A-G/G-(exon)-3'.The upstream and downstream splice sites in nuclear RNA molecules presumably must be brought together for RNA splicing to occur. This could be achieved by intramolecular base pairing, but no appropriate complementary structures have been found reproducibly in the vicinity of splice sites (10). We propose an alternative mechanism in which upstream and downstream splicing sites form base pairs with another RNA molecule which could be a structural component of the splicing enzyme(s). There may be an example comparable to this proposed splicing system in RNase P of Escherichia coli, which is a site-specific RNase with an essential RNA component (I1).THE MODEL We considered that such an effector RNA might be found among the discrete; stable, small nuclear RNAs (snRNAs) that are ubiquitous in eukaryotic cells (12). Sequences have been reported for several of these (13-17). When we compared the sequence of U-i snRNA from rat hepatoma (13) with sequences of the optimal splice site (Fig. 1), we discovered that the first 10 nucleotides after the U-1 RNA cap are perfectly complementary to the 9 nucleotides of the optimal upstream site and to the tetranucleotide C-A-G/G from the optimal downstream site. The next 11 nucleotides of U-I are purine-rich and can be aligned with the pyrimidine-rich segment of the downstream site. This striking complementarity with splicing sites is not found in any of the other snRNAs now sequenced, which are U-2 (14), 4.5S (15), and adenovirus VA-I RNAs (16,17). We therefore propose that U-1 snRNA in the RNA splicing complex forms base pairs with both ends of an intron in a large nuclear RNA molecule as shown in Fig. 2, bringing

show abstract

Section: '-T T T T(t)t T T T T T Ct Tncamentioning

confidence: 99%

A mechanism for RNA splicing.

Rogers¹,

Wall²

1980

Proc. Natl. Acad. Sci. U.S.A.

475

162

View full text Add to dashboard Cite

show abstract

“…A correlation analysis of available sequences of eukaryotic DNAs and mRNAs (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26) as well as of some viral DNAs (27)(28)(29)(30)(31)(32)(33)(34)(35) that are known to be folded in chromatin-like structures (36,37) shows that some of the dinucleotides indeed exhibit this periodicity, although it is well hidden. The period is estimated to be 10.5 + 0.2 bases, which is consistent with recent measurements and estimations of the pitch of DNA in dilute solution and in chromatin (6,38).…”

mentioning

confidence: 99%

The pitch of chromatin DNA is reflected in its nucleotide sequence.

Trifonov¹,

Sussman²

1980

Proc. Natl. Acad. Sci. U.S.A.

525

299

View full text Add to dashboard Cite

show abstract

“…The 900-bp insert of p167KR1 (18), a K light-chain cDNA clone deriving from the murine plasmacytoma MOPC167, was used under standard hybridization conditions to identify K clones. -y heavy-chain clones were identified under standard hybridization conditions with probe prepared from pH21.1 (25), a plasmid containing sequences encoding amino acids 287 to 439 of the constant region of the murine -yl heavy chain deriving from the plasmacytoma MOPC21. A third probe, pS107V1 (7,8), containing an entire VH region, including a D region and part of a J region, was further used under conditions of low stringency to identify cDNA clones containing V-region heavy-chain sequences.…”

Section: Methodsmentioning

confidence: 99%

A new means of inducibly inactivating a cellular protein.

Carlson¹

1988

Mol. Cell. Biol.

View full text Add to dashboard Cite

This paper presents a general means of eliminating the function of a single protein without relying on genetic alterations in its structure or level of synthesis. The strategy is based on the inducible cellular expression of neutralizing antibody to inactivate the protein selectively. The feasibility of this approach is illustrated by using alcohol dehydrogenase I (ADH I) in Saccharomyces cerevisiae as a model. Heavy-and light-chain cDNAs were isolated from a hybridoma secreting an antibody which neutralizes yeast ADH I. The cDNAs were characterized with respect to their length and identity, their signal sequences were removed, and synthetic translation initiation codons were joined to them. These truncated sequences were then inserted into an inducible expression vector and shown to be expressed as stable heavy and light chains, which assemble and bind antigen. The sequences were introduced into yeast mutants containing different levels of ADH activity, and evidence is provided that the antibodies produce limited neutralization of enzyme activity in vivo. In principle, the approach can be used for any cell type in which functional antibody can be inducibly expressed. There is ample precedent from microinjection experiments for the ability of antibodies to cause phenotypic effects in vivo (see, e.g., references 9 and 26). In one such experiment, antibodies were shown to be able to react with antigen in the cytoplasm for at least 6 h (30). Other methods of eliminating the activity of specific proteins, including one based on the use of antisense RNA, have recently been developed (15) or proposed (12), although the extent of their utility is not yet clear.As a model system in which to test the feasibility of this approach, the yeast enzyme alcohol dehydrogenase I (ADH I) was chosen because of the extent of its physical (19) and genetic (6, 31) characterization, its cytoplasmic location, the availability of a convenient in vitro assay for its activity, and, above all, the ability to select both for and against its activity. Three monoclonal antibodies which potently neutralize ADH activity in a yeast extract and show no detectable cross-reactivity with other cell components are described in the accompanying paper (5).The present article describes the isolation of heavy-and light-chain cDNA clones encoding one of these antibodies, the removal of their signal sequence coding regions, and the addition of synthetic translation initiation codons to their 5' ends. These truncated sequences are then inserted into a single expression vector in such a manner that both are transcribed coordinately under inducible control in the yeast Saccharomyces cerevisiae. Stable heavy-and light-chain polypeptides are synthesized, assemble, and bind antigen. Finally, evidence is presented that limited in vivo neutralization occurs upon induction of antibody synthesis. MATERIALS AND METHODSConstruction and screening of a hybridoma cDNA library. RNA was extracted from hybridoma cells by extraction in guanidine hydrochloride, and poly(A)+ RN...

show abstract

Sequence analysis of cloned cDNA encoding part of an immunoglobulin heavy chain

Cited by 39 publications

References 43 publications

A mechanism for RNA splicing.

A mechanism for RNA splicing.

The pitch of chromatin DNA is reflected in its nucleotide sequence.

A new means of inducibly inactivating a cellular protein.

Contact Info

Product

Resources

About