Aortic preferentially expressed gene (APEG)-1 is a 1.4-kilobase pair (kb) mRNA expressed in vascular smooth muscle cells and is down-regulated by vascular injury. An APEG-1 5-end cDNA probe identified three additional isoforms. The 9-kb striated preferentially expressed gene (SPEG)␣ and the 11-kb SPEG␤ were found in skeletal muscle and heart. The 4-kb brain preferentially expressed gene was detected in the brain and aorta. We report here cloning of the 11-kb SPEG␤ cDNA. SPEG␤ encodes a 355-kDa protein that contains two serine/threonine kinase domains and is homologous to proteins of the myosin light chain kinase family. At least one kinase domain is active and capable of autophosphorylation. In the genome, all four isoforms share the middle three of the five exons of APEG-1, and they differ from each other by using different 5-and 3-ends and alternative splicing. We show that the expression of SPEG␣ and SPEG␤ is developmentally regulated in the striated muscle during C2C12 myoblast to myotube differentiation in vitro and cardiomyocyte maturation in vivo. This developmental regulation suggests that both SPEG␣ and SPEG␤ can serve as sensitive markers for striated muscle differentiation and that they may be important for adult striated muscle function.

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155Fifty-five haemin-requiring mutants were isolated from haemin-permeable mutants. According to their growth responses to haem precursors and their patterns of porphyrin accumulation, the 55 mutants fell into three groups which were judged to have defects in 5-aminolaevulinate dehydratase, ferrochelatase, and uroporphyrinogen I11 cosynthase or uroporphyrinogen decarboxylase. In mutants of the group deficient in 5-aminolaevulinate dehydratase, the mutations were adjacent to lac, and evidence is presented that the mutations were in hemB and were commonly deletions extending into proC. I N T R O D U C T I O NEscherichia coli K12 is impermeable to haemin under normal cultural conditions (SMrman et al., 1968), and genetic analysis of haem biosynthesis has depended on indirect methods. McConville & Charles (1975, 19798) described mutants of E. coli IS12 which are permeable to haemin. The present paper describes the isolation and study of haeminrequiring mutants derived from haemin-permeable mutants. M E T H O D SUnless stated otherwise, the methods were those used by McConville & Charles (1979~). Media. Glucose minimal medium (GM) was medium E of Vogel & Bonner (1956) supplemented with thiamin (5 mg 1-l) and glucose (5 g 1-l). Glucose minimal agar (GMA) was GM containing Difco Bacto agar (13 g P ) , autoclaved separately. When necessary, the glucose in GM was replaced by other sugars (2 g 1-l). Solutions of amino acids and other growth factors were sterilized by membrane filtration and aseptically added to minimal medium to give a final concentration of 40 mg l-l, except isoleucine and valine were added at 12 and 28 mgl-l, respectively, and 5-aminolaevulinic acid (5-ALA) at 1 mg 1-' . TNM contained (g 1-l): NaCl, 4; Difco Bacto nutrient broth, 8 ; MgS04. 7H20, 2.47. TNAM was TNM autoclaved with agar (13 g I-').Stock solutions of haemin (type 111, Sigma) and protoporphyrin IX (PP9; Calbiochem) were prepared by dissolving 0.2 g in 5 ml Tween 80 and slowly diluting to 50 ml with sterile distilled water; a few drops of alkali were added to give clear solutions. The procedure gave sterile solutions which were stored at -18°C and added aseptically to give concentrations of 25 mg 1-1 in TN media and 10 mg 1-1 in GM media.Genetical analysis. The transducing phage was Plkc; 12 min adsorption were allowed. Gene symbols and references to the E. culi linkage map follow Bachmann e f al. (1976). Details of the strains are given in Table 1.Assay ofporphyrins accumulated in cultures. Bacteria were inoculated into two 2 1 flasks containing glucose minimal medium (400 ml) plus sodium thiosulphate (10 mg 1-l) and 5-ALA (50 mg I-l); one flask contained haemin (10 mg 1-l). Proline was added if required. Incubation was for 48 h at 37 "C in an orbital incubator. Bacteria were then washed and resuspended in 10 ml of glass-distilled water, and a 1 ml sample was removed for drying and weighing. Porphyrins were extracted from the supernatant medium and bacteria by the method of Falk (1964). Coproporphyrin and protoporphyrin were extracted fr...

47As the first stage in investigating the genetic basis of natural variation in Escherichia coli, the gene(s) conferring the ability to use sucrose as a carbon and energy source (given the symbol sac+) was transferred from a wild strain to ~1 2 , which does not use sucrose. The sac+ region was transferred by two different methods. On both occasions it took a chromosomal location at minute 50-5 on the linkage map, between aroC and supN, in the region of the dsd genes, which confer the ability to use D-serine as a carbon and energy source. When the sac+ region was present in the K 12 chromosome the bacteria were unable to use D-serine as a carbon and energy source. In F'sac+/dsd+ diploids, the dsd+ genes were similarly not expressed. Strain ~1 2 ( s a c + ) bacteria were sensitive to inhibition by D-serine; they mutated to D-serine resistance with much greater frequency than did a dsd mutant of ~1 2 .Such bacteria also mutated frequently to use raffinose. Strain K l 2 ( S U C + ) bacteria did not utilize sucrose when they carried a mutation affecting the phosphotransferase system.

SUMMARYThis report shows that some auxotrophic mutants of Escherichia coli can grow on a minimal medium without growth factors when the gas phase is supplemented with carbon dioxide. Mutants which respond to CO, are called CO, mutants. The CO, mutants of E. coli resemble those already known in Neurospora. When the gas phase is not supplemented with CO, most of the mutants respond to other specific growth factors such as arginine, uracil, adenine, succinate or isoleucine + valine, depending upon the locus of their mutation. One mutant is an obligate CO, mutant. The CO, effects shown by these mutants are discussed in relation to the general problem of CO, effects in micro-organisms.