Three unusually large myofibrillar proteins (Mn, >500,000) are present in major amounts in the striated muscle of various species. The molecular properties and localization of two of these proteins (M,, M1 X 106), named "titin," have been reported recently [Wang, K., McClure, J. & Tu, A. (1979) Proc. NatL Acad. Sci. USA 76,3698-37021. We have now purified the third protein (M,, s5-6 X 105), designated "band 3," from rabbit psoas myofibrils. Band 3 is distinct chemically and immunochemically from titin and myosin heavy chain.Immunofluorescent localization studies of band 3 in glycerinated rabbit psoas myofibrils indicated a biphasic distribution.In sarcomeres 1.8-3.7 gm long, band 3 is localized predominately on the N2 line in the phase-contrast lucent region of the I band. Quantitative analysis of light micrographs confirmed the unique behavior of N2 lines described in early electron microscopic studies. As the sarcomeres lengthen, the N2 line moves away from both the Z line and the M line, maintaining the same roportional distance. In very short sarcomeres (1.5-1.8 pm), band 3 is found mainly in the A band, suggesting that either the N2 line moves into the A band or that band 3 has dissociated from the N2 line which remains near the Z line. We conclude that band 3 is an N2 line component of rabbit skeletal myofibrils.In vertebrate striated muscle, two prominent electron-dense bands, Z and M lines, interconnect thin and thick filaments, respectively, to maintain proper lateral and longitudinal register during contraction. Furthermore, Z lines serve as a link for the transmission of tension along myofibrils and for the coordination of movement of adjacent myofibrils (for reviews, see refs. 1-4). In addition to Z and M lines, two faintly stained transverse structures, N1 and N2 lines, have been located within the I band by electron microscopy (5-9). These lines frequently appear as rows of bead-like thickenings along thin filaments on either side of and parallel to Z lines. The Ni line is narrow (;0.05 Am) and fixed in position, 0.1-0.2 ,um from the Z line center; in contrast, the N2 line is wider (up to 0.15 ,m) and varies in position with sarcomere length (5, 6). For reasons still unclear, these lines are not always observable by electron microscopy in any given striated muscle. It has been suggested that their location in the I band could mean that they are involved in the regulation or maintenance of the changing spatial arrangement of thin filaments from a square lattice at the Z line to a hexagonal array near the A-I junction (6, 9). The protein constituents of either line are completely unknown.The presence of three unusually large, major myofibrillar proteins in a wide range of striated muscle has been reported (10). The molecular properties and localization of two of these proteins (Mr, _u1.0 -X 106), designated "titin," in chicken myofibrils have been described (10-12). We have now purified the third protein, designated "band 3," from rabbit psoas muscle (Mr,. X 105) and prepared specific an...

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We have cloned a DNA complementary to the messenger RNA encoding the precursor of ornithine transcarbamylase from rat liver. This complementary DNA contains the entire protein coding region of 1062 nucleotides and 86 nucleotides of 5'- and 298 nucleotides of 3'-untranslated sequences. The predicted amino acid sequence has been confirmed by extensive protein sequence data. The mature rat enzyme contains the same number of amino acid residues (322) as the human enzyme and their amino acid sequences are 93% homologous. The rat and human amino-terminal leader sequences of 32 amino acids, on the other hand, are only 69% homologous. The rat leader contains no acidic and seven basic residues compared to four basic residues found in the human leader. There is complete sequence homology (residues 58-62) among the ornithine and aspartate transcarbamylases from E. coli and the rat and human ornithine transcarbamylases at the carbamyl phosphate binding site. Finally, a cysteine containing hexapeptide (residues 268-273), the putative ornithine binding site in Streptococcus faecalis, Streptococcus faecium, and bovine transcarbamylases, is completely conserved among the two E. coli and the two mammalian transcarbamylases.

Compensatory mutations have been constructed which demonstrate that P8 and P6, two of nine proposed base-pairing interactions characteristic of group I introns, exist within the folded structure of the Tetrahymena thermophila rRNA intervening sequence, and that these secondary structure elements are important for splicing in E. coli and self-splicing in vitro. Two-base mutations in the 5' and 3' segments of P8 are predicted to disrupt P8 and a strong splicing-defective phenotype is observed in each case. A compensatory four-base mutation in P8 is predicted to restore pairing, and results in the restoration of splicing activity to nearly wild type levels. Thus, we conclude that P8 exists and is essential for splicing. In contrast to the strong phenotypes generally exhibited by mutations which disrupt RNA secondary structure, a two-base mutation in L8, the loop between P8[5'] and P8[3'], results in only a slight decrease in splicing activity. We also tested P6, a pairing which is proposed to consist of only two base-pairs in this intron. A two-base mutation in P6[3'] reduces splicing activity to a greater extent than does a two-base mutation in P6[5']. Comparison of the activities of these mutants and a compensatory P6 four-base mutant support the existence of P6, and suggest that the P6 pairing may be particularly important in the exon ligation step of splicing.

We have developed conditions for efficient cDNA cloning of nanogram amounts of purified mRNAs coding for cystathionine ,B-synthase [L-serine hydro-lyase (adding homocysteine), EC 4.2.1.22] and for the cytosolic precursors of mitochondrial ornithine transcarbamylase (carbamoylphosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3) and the (3 subunit of propionyl-CoA carboxylase [propanoyl-CoA: carbon-dioxide ligase (ADP-forming), EC 6.4.1.3]. The three mRNAs, prepared by sequential immunoselection from the same batch of rat liver polysomes, were pooled (20 ng each), and cDNA was synthesized by using avian reverse transcriptase. The second DNA strand was prepared by "nick-translation repair" of the cDNAimRNA hybrid with RNase H, polymerase I, and DNA ligase from Escherchia coil. The double-stranded (ds) DNA was tailed with deoxycytidine residues, annealed with Pst I-cut/dG-tailed pBR322, and used to transform E. coli. The library generated by this three-step procedure contained 5000 independent colonies. A 550-basepair (bp) cDNA clone of the ,B subunit of propionyl-CoA carboxylase was detected by hybrid-selected translation; it was then used to screen the library for longer cDNAs. Two hybridizing cDNAs, 1200 and 1000 bp long with a 200-bp overlap, representing together a full-length copy of the coding region and 446 bp of 3' untranslated sequence, were recovered.Each plasmid mapped to the region ql3.3-*q22 of human chromosome 3. Cystathionine (-synthase clones were obtained by screening the library with a single-stranded [32P]cDNA prepared directly from the highly purified synthase mRNA by reverse transcriptase. The longest hybridizing cDNA of 1700 bp was used in hybrid-selected translation and detected a polypeptide of 63 kDa, identical in size to rat liver synthase. In situ hybridization of this cDNA to q22 of human chromosome 21 confirmed two previous tentative assignments of the synthase locus to this chromosome.We have demonstrated (1, 2) that low-abundance mRNAs can be enriched to near homogeneity, using the method of polysome immunopurification with polyclonal antibodies to rat liver proteins. The quantity of purified mRNA produced by this method is quite small, however, being typically less than 20 ng/10 g of liver. Therefore, our next goal was to show that the minute amounts of mRNA obtained in this manner could be used to generate cDNA libraries containing long transcripts of these messages with intact 5' ends. The generally used conditions for cDNA cloning have remained basically the same as those developed initially for globin mRNA-an abundant and rather short RNA. The first cDNA strand usually serves as both a primer and a template for the poorly controlled synthesis of the second cDNA strand. S1nuclease digestion of the single-stranded DNA loop prior to insertion of the double-stranded (ds) DNA into plasmid invariably removes portions of the 5'-end sequence. These conditions yield cloned cDNAs that are heavily biased for the 3' end of the mRNA. Furthermore, the efficiency of the procedure i...