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A TPS-reactive protein fragment from a human colon adenocarcinoma cell line was purified to electrophoretic homogeneity. N-terminal sequence analysis of the purified 13 kDa protein fragment demonstrated that the component was a fragment of human cytokeratin 18. The M3 monoclonal antibody (detector antibody in the TPS assay) was applied for screening of an expression library. The M3-reactive phage clones were subcloned and PCR amplified. DNA-sequence analysis revealed it to contain a nucleotide fragment corresponding to human cytokeratin 18. Smaller fragment, engineered by PCR and expressed as fusion proteins, demonstrated that the M3 epitope is localized to human cytokeratin 18, amino acid residues 322-340.

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Tissue-polypeptide-specific antigen (TPS) from the human colon adenocarcinoma cell line WiDr was purified using the monoclonal antibody M3 as a probe. Upon SDS/polyacrylamide gradient gel electrophoresis, several TPS-positive bands were detected (corresponding to 13 kDa, 22 kDa and a doublet at 42 kDa). The 13-kDa moiety was purified about 30,000-fold by a 5-step protocol. The electro-phoretically homogeneous component was obtained in a 7% yield of the total TPS activity of the crude extract. N-terminal sequence analysis showed the presence of an N-terminally truncated molecule and identified the 13-kDa TPS component as a fragment of human cytokeratin 18, with a major from starting at position 284 of the parent molecule. Laser-desorption mass spectrometry showed the presence of one major component with a molecular mass corresponding to a C-terminal end close to position 396 (which gives 12776 Da for the form with non-truncated N-terminus). The M3 antibody was also used to screen a human prostate cDNA lambda gt11 library. Four identical phage clones were detected, each producing a fusion protein with beta-galactosidase and the M3-positive component. PCR amplification showed the presence of an approximately 1200-bp insert, and sequence analysis revealed it to contain a 996-nucleotide fragment corresponding to residues 103-429 of human cytokeratin 18 (plus a non-coding human desmin artifact fragment). Smaller fragments, engineered by PCR and expressed as fusion proteins using the pET3xc vector in Escherichia coli, showed that the M3 epitope is localized to cytokeratin 18, residues 322-340. Two other TPS-active monoclonal antibodies were localized to cytokeratin 18 with similar techniques, ascribing an epitope (to M21) to residues 414-429 and another (to M24) to residues 139-297. Combined, the results demonstrate that TPS reactivity is derived from specific epitopes of human cytokeratin 18.

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Normal human sera contain one or several factors cytotoxic for normal guinea pig thymocytes, and when serum is precipitated with ammonium sulphate (60% saturated) and the precipitate dissolved and dialyzed, the activity is preserved. Gel chromatography with Sephadex G-150 and Sepharose CL-6B indicated a molecular weight of approximately 900,000 daltons. The active fractions contained a high amount of IgM according to single radial immunodiffusion and two-dimensional gel electrophoresis. Quantitation of the IgM band in one-dimensional gel electrophoresis preparations by gel scanning indicated that IgM accounted for 65% of the eluted proteins in active fractions. Purified human IgM from myeloma patients eluted as the active factor during gel chromatography. Elimination of IgM from serum by affinity chromatography eliminated the cytotoxic activity. The serum could also be inactivated by heating. The mixing of IgM-depleted serum with either polyclonal IgM or heat-inactivated serum restored the activity. Thus, the cytotoxic activity is due to IgM antibodies plus a heat-labile component (presumably complement). The presence of the cytotoxic activity in autologous (guinea pig) serum was recently demonstrated. The possible functional role of these antibodies in the elimination process of a large number of cortical thymocytes is suggested.

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The two giant secretory proteins, sp-Ia and sp-Ib, in salivary-gland cells of the larva of the fly Chironomus tentans, were isolated by preparative gel electrophoresis and characterized chemically. Their amino acid compositions are dominated by polar amino acids, with about 30% of basic amino acid residues. Crossed immunoelectrophoresis of sp-Ia and sp-Ib provided evidence that they share antigenic determinants. They also have major methionine-containing tryptic peptides in common. CNBr cleavage of sp-Ib gives a small number of low-Mr fragments, indicating that this protein has a repetitive structure.

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Balbiani rings (BR), giant puffs in Chironomus larval salivary glands, code for giant secretory proteins. As shown earlier, the normally dominant BR2 is turned off with its putative translation product during exposure of larvae to compounds that diminish the stores of P(i). A BR6 develops from a compact chromosome band, and a new giant protein appears in the secretion as the major component. We have determined the sequence of cloned DNA fragments representative for large parts of BR1 and BR2 (normally active) and the inducible BR6. There is an excess of positive charges and high contents of serine/threonine in the coded amino acid composition for the BR1 and BR2 sequences. The coded amino acid sequence for the BR6 clone shares homologies with the others but has an excess of negative charges and lacks serine/threonine. This suggested that the P(i) effects observed earlier could be related to differences in phosphorylation between the normal proteins and the BR6 product. This could be confirmed by measurements of phosphorylation, which occurs in the normal giant proteins mainly at seryl residues. P export with giant secretory protein is normally quantitatively important. Thus, BR6 activation should decrease P loss when P(i) pools are lowered because of inducer action.

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We have investigated whether Balbian ring derived mRNA (75S RNA) of Chironomus salivary glands spreads to the different parts of the cytoplasm before or after attachment to the rough endoplasmic reticulum. Glands were fixed after in vivo administration of tritiated uridine and cytidine and the appearance of labelled RNA fractions in microdissected cytoplasm was measured. We chose central cytoplasm rich in rough endoplasmic reticulum (endoplasm) and peripheral cytoplasm with a low content of reticulum (basal zone). A peripheral spread of the mRNA on reticulum membranes would have moved the labelled RNA outwards with time. We found, however, a redistribution in the opposite direction, suggesting that the mRNA spreads over the whole cytoplasm before it attaches to the reticulum membranes. A similar redistribution occurs for the ribosomal subunits.

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The 75S RNA originating in the large Balbiani rings 1 and 2 (BRI and 2) was isolated and used for in vitro translation in the mRNA dependent reticulocyte lystate. Conditions (K+-concentration, temperature, time etc). were optimized for obtaining translation products of maximal size. Polypeptide chains up to about 500,000 D were obtained but no complete translation products. Trypic fingerprints were performed on the in vitro products as well as on the secretory protein components nos. I and II+III labelled with 35S-methionine. There was a large degree of correspondence between the fingerprint of the in vitro product and that of component I but less to that of component II+III. The results suggest that 75S RNA with an origin in the BR1 and BR2 codes for the giant secretory protein component I.

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The main secretory protein fractions from Chironomus tentans have been investigated with particular emphasis on the dominant fraction, component 1, here designated I (Grossbach, 1969). This polypeptide was suggested to be the translatory product of 75SS RNA from Balbiani ring 2 (BR2) because of its size and quantitative prominence. Its molecular weight was estimated by gel filtration in 8 M urea at 850,000 + 101,000 D. During short pulses with radioactive amino acids a large fraction of the label was found in a population of polypeptide chains suggestive of molecules continuously growing to the size of compoenet I. Populations of nascent large protein chains of similar size distribution were dominant in the polysomes and constituted the only population present in the largest polysomes, known to contain 75S RNA from BR2 (and BR1) as predominant or only component (Daneholt et al., 1977; Wieslander and Daneholt, 1977). These data indicate strongly that the large size of component I is not a result of posttranslational modifications. No sequence similarities, using limited proteolysis, were found between component I and component II, both of which have been considered to the BR2 products. There was, furthermore, no detectable immunological identity between component I and smaller secretory protein fractions. The data support Grossbach's and Daneholt's suggestion that component I is closely related to the primary translation product of 75S RNA from the large Balbiani rings.

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The distribution of monodisperse high molecular weight RNA (38, 30, 28, 23, and 18S RNA) was studied in the salivary gland cells of Chironomus tentans. RNA labeled in vitro and in vivo with tritiated cytidine and uridine was isolated from microdissected nucleoli, chromosomes, nuclear sap, and cytoplasm and analyzed by electrophoresis on agarose-acrylamide composite gels. As shown earlier, the nucleoli contain labeled 38, 30, and 23S RNA. In the chromosomes, labeled 18S RNA was found in addition to the 30 and 23S RNA previously reported. The nuclear sap contains labeled 30 and 18S RNA, and the cytoplasm labeled 28 and 18S RNA. On the basis of the present and earlier analyses, it was concluded that the chromosomal monodisperse high molecular weight RNA fractions (a) show a genuine chromosomal localization and are not due to unspecific contamination, (b) are not artefacts caused by in vitro conditions, but are present also in vivo, and (c) are very likely related to nucleolar and cytoplasmic (pre)ribosomal RNA. The 30 and 23S RNA components are likely to be precursors to 28 and 18S ribosomal RNA. The order of appearance of the monodisperse high molecular weight RNA fractions in the nucleus is in turn and order: (a) nucleolus, (b) chromosomes, and (c) nuclear sap. Since both 23 and 18S RNA are present in the chromosomes, the conversion to 18S RNA may take place there. On the other hand, 30S RNA is only found in the nucleus while 28S RNA can only be detected in the cytoplasm, suggesting that this conversion takes place in connection with the exit of the molecule from the nucleus.

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