Most eucaryotic mRNAs are polyadenylated. In higher eucaryotes, the sequence AATAAA is located 7 to 30 base pairs (bp) upstream from the site of processing and polyadenylation and is a critical part of the signal for processing and polyadenylation. Efficient cleavage and polyadenylation also require sequences downstream of polyadenylation sites. The herpes simplex virus type 1 thymidine kinase (tk) gene contains two copies of the AATAAA hexanucleotide and a GT box (18 of 19 consecutive residues are G or T) previously shown to be required for efficient processing and polyadenylation of tk mRNA (C. N. Cole and T. P. Stacy, Mol. Cell. Biol., 5:2104-2113). To define further the sequence requirements for efficient polyadenylation, we prepared linker scanning, internal deletion, and small insertion mutations in the polyadenylation region of the tk gene. These mutations were analyzed by S1 nuclease protection analysis of cytoplasmic RNA isolated from transfected Cos-1 monkey kidney cells. When the proximal AATAAA was deleted, no tk mRNA polyadenylated in the normal region was detected, whereas replacement of the second AATAAA with an XbaI linker had no effect on polyadenylation. When various portions of the GT box were replaced with linker, the amount of tk mRNA produced was reduced to 23 to 82% of the normal amount, but polyadenylation in the normal region was never abolished. Thus, no single portion of the GT box was absolutely required. In some cases, extended transcripts, polyadenylated at a cryptic site within pBR322, were detected. A spacing of 6 bp between AATAAA and the GT box was too short for efficient processing and polyadenylation. A spacing of 30 bp appeared to work almost as efficiently as did the wild-type spacing of 18 bp. Taken together, these results indicate that efficient polyadenylation requires both AATAAA and downstream GT-rich sequences. In addition, processing and polyadenylation are affected both qualitatively and quantitatively by sequences at polyadenylation sites and at more distant locations.
Thy-1 was originally defined in mice as a cell-surface alloantigen of thymus and brain with two allelic forms, Thy-1.1 and Thy-1.2 (ref. 1). Subsequently, the Thy-1.1 alloantigenic determinant was identified in rats. In both species, Thy-1 is present in large amounts on thymus and brain cells and in smaller quantities on fibroblasts, epidermal cells, mammary glands and immature skeletal muscle. In many of these tissues the level of Thy-1 expression changes dramatically during cell differentiation. The molecules expressing the Thy-1 antigenic determinant have been isolated from rat and mouse brain cells and have been shown to have a molecular weight of 17,500 (ref. 8). One-third of the Thy-1 molecule is carbohydrate and the remainder is a polypeptide of 111 amino acids whose sequence has been fully determined. We report here the isolation and characterization of a cDNA clone encoding the rat thymus Thy-1 antigen but find that the DNA sequence ends prematurely at a position corresponding to amino acid 103. It appears to be a complete transcript, however, as the last codon is followed directly by a poly(A) tract.
(6,7,11,24,35,36,41) [37,40]), RNAs produced using alternate poly(A) sites contain the same protein-coding region. The function of these different 3'-flanking regions is unknown.Plasmids and viruses containing two poly(A) signals have been constructed (9,12,13,18,29,31). In these studies, both polyadenylation signals were used under at least some conditions. When a signal was present twice, the promoterproximal signal was usually used more. In most cases, poly(A) site choice was not the major emphasis of the study and other aspects of RNA metabolism (turnover, time of appearance, etc.) were not assayed. * Corresponding author.In this study we have addressed several unresolved questions about poly(A) site selection in the presence of multiple poly(A) signals. The simian virus 40 (SV40) late poly(A) signal (SVL), the herpes simplex virus (HSV) thymidine kinase poly(A) signal (TK), and an 88-base-pair (bp) AA TAAA-containing DNA fragment from the SV40 early region (88 signal) were inserted in single or multiple copies 3' to the HSV tk-coding region. The quantity and 3'-end structure of RNAs produced from these constructs during transient expression in Cos-1 monkey kidney cells were assayed by S1 nuclease protection. Although all signals in each construct were used, increasing the number of poly(A) signals from one to four did not significantly affect the amount of RNA produced, regardless of how much RNA was produced from a construct with a single copy of the signal. Increasing the distance between two signals favored the use of the 5' signal, while changing the distance between the promoter and the first signal had no effect on signal use. In concert with data from experiments assaying various aspects of RNA metabolism, these data are inconsistent with a number of models of poly(A) signal recognition. A model involving scanning from the point of transcription is consistent with our data. MATERIALS AND METHODSPlasmids, plasmid DNA, and bacteria. Bacterial growth and transfection (21), minilysate DNA preparation and analysis (4), and large-scale plasmid purification (10) were performed by using standard techniques. All plasmids were propagated in Escherichia coli HB101.Plasmid construction. Plasmid pTK, (Fig. 1 and 2) is the same as pTK205R/SV010 described by Cole and Stacy (6). It was produced by BAL 31 nuclease resection of pTK2/SVO10 (5) and contains the tk-coding region, both sequence elements of the polyadenylation signal, and 62 bp 3' to the poly(A) site. pTK206RISVO10 (6, 41) contains the tk-coding 4829
Thy-1 is a differentiation marker expressed predominantly on thymocytes, T cells and brain tissue. Its presence on murine peripheral T cells but not B cells has long been used to distinguish between these two populations of lymphocytes. Although analogues of Thy-1 have been described in several mammalian species, its tissue distribution in different species varies widely, precluding its use as T-cell-specific marker. The Thy-1 molecule is a cell-surface glycoprotein of relative molecular mass 18,000, one-third of which represents carbohydrate; the protein moieties of the rat and murine Thy-1 molecules have been sequenced and found to consist of 111 and 112 amino acids, respectively. An unusual aspect of Thy-1 is the apparent absence of a hydrophobic segment comparable to that observed in other membrane glycoproteins which would allow integration of Thy-1 within the membrane lipid bilayer. This has prompted speculation that Thy-1 is anchored to the cell surface by some other hydrophobic component such as glycolipid. Here we report the structure of thy-1 complementary DNA and genomic clones and describe the exon-intron organization of the gene. More importantly, our data indicate that Thy-1 is initially synthesized as a molecule of 142 amino acids, 31 amino acids longer at the carboxyl end than the Thy-1 molecule isolated and characterized by Campbell et al. An extremely hydrophobic region of 20 amino acids lies within this 31-amino acid stretch and may represent the transmembrane segment responsible for anchoring Thy-1 to the cell membrane.
The mode of integration of the glycoprotein thy-1 within the cell membrane has been controversial due to an apparent lack of a transmembrane hydrophobic segment. Rat and mouse complementary DNA and genomic clones encoding the thy-1 molecule have been isolated and sequenced. These studies have enabled us to determine the intron-exon organization of the thy-1 gene. Furthermore, they have revealed the existence of a sequence which would encode an extra segment (31 amino acids) at the carboxyl terminus of the thy-1 molecule. These extra amino acids include a 20-amino acid hydrophobic segment which may be responsible for integration of thy-1 within the plasma membrane.
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