1. Catechol was metabolized through 2-hydroxymuconic semialdehyde by cell-free extracts of benzoate-grown Azotobacter strains. Some properties of catechol 2,3 oxygenase preparations from Azotobacter vinelandii 206 are described.2 . Two different enzymatic activities able to attack 2-hydroxymuconic semialdehyde have been found in crude extracts from benzoate-grown cells ; one catalyses a hydrolytic release of formate from the semialdehyde and the other a dehydrogenation of this compound to 4-oxalocrotonate. However, the low, non-inducible levels of 2-hydroxymuconic semialdehyde hydrolase activity appear negligible for metabolic purposes and the semialdehyde seems to be dissimilated almost exclusively via 4-oxalocrotonate, by the action of a NAD+-dependent dehydrogenase, in Azotobacter strains.3, A tautomerase activity responsible for the interconversion of the enol and keto forms of 4-oxalocrotonic acid was found in extracts from benzoate-grown cells.4. 4-Oxalocrotonate was stoicheiometrically converted to CO, and 4-hydroxy-2-oxovalerate by a partially purified extract, with the transient formation of a compound that appears to be 2-oxopent-4-enoic acid. The 4-oxalocrotonate decarboxylase activity was stimulated by Mg2+ or Mn2+ ions and was inhibited by EDTA.5. Cell-free extracts from Azotobacter strains converted synthetic 4-hydroxy-2-oxovalerate to acetaldehyde and pyruvate.6. A reaction sequence, termed the 4-oxalocrotonate pathway, for the dissimilation of catechol to acetaldehyde and pyruvate by Azotobacter species is presented. All the enzymes operative in this pathway were inducible, except the 4-hydroxy-2-oxovalerate aldolase. 7. The findings described here are discussed in connection with the two previously reported meta cleavage pathways for the oxidation of catechol in Pseudomonas strains.

The rat serum albumin gene has been isolated from a recombinant library containing the entire rat genome cloned in the X phage Charon 4A. Preliminary R-loop and restriction analysis has revealed that this gene is split into at least 14 fragments (exons) by 13 intervening sequences (introns), and that it occupies a minimum of 14.5 kilobases of genomic DNA.Recent advances in recombinant DNA technology have made it possible to obtain virtually any desired single-copy genomic sequence in cloned form, provided an appropriate probe is available. We have used these techniques to isolate the rat serum albumin gene. Serum albumin synthesis is one of the major characteristics of vertebrate liver. Observation of the activity and state of this gene during development and in adult tissues should be informative as to the process of terminal differentiation. Albumin synthesis is essentially constitutive, but does respond significantly to a variety of stimuli (1). It is also expressed to variable extents in different hepatoma cell lines (2). The availability of cloned albumin genomic DNA will greatly facilitate the study of this variable expression, particularly at the level of transcript processing.Determination of the sequence organization of the albumin gene is also of interest, especially with regard to the disposition of repetitive elements and intervening sequences. Although regulatory (3) and evolutionary (4) significance has been postulated, the functional role, if any, of these striking features of eukaryotic genomes remains unknown. The comparative studies that will be possible as other genes are extracted from the rat and related species can be expected to provide considerable insight into this fascinating problem. MATERIALS AND METHODSRat Genome Library. High molecular weight liver DNA was extracted from an adult male Sprague-Dawley rat (Simonsen Labs, Gilroy, CA) by the method of Blin and Stafford (5) and aliquots were digested with EcoRI (Boehringer Mannheim) under conditions adjusted to cleave either one-third or one-fifth of the EcoRI sites in an equivalent amount of bacteriophage X DNA. The fragments resulting from this partial digestion were sedimented through a 10-30% sucrose gradient; the material between 10 and 20 kilobases (kb) was recovered by ethanol precipitation. A sample of this rat DNA (2.5 ,g) was ligated with 8.5 ,ig of a preparation of Charon 4A "cloning fragments" (6, 7). This recombinant DNA was packaged in vitro by using extracts from defective X lysogens provided by N. Sternberg (6). The method used was that of Hohn and Murray (8). Approximately 2,000,000 independent clones were obtained. The library was amplified 100,000-fold by subconfluent plating on Escherichia coli strain DP5OSupF (9).cDNA Clones. cDNA was synthesized from purified albumin mRNA as described (10). This cDNA contained a small amount of full-length material and had a number average size of approximately 1000 nucleotides. It was rendered double-stranded by sequential treatment with E. coli DNA polymerase I and S1 nuc...

1. Pseudomonas arvilla mt-2 grows a t the expense of benzoate, m-toluate (3-methylbenzoate) and p-toluate (4-methylbenzoate), but not o-toluate (2-methylbenzoate). Under various conditions compounds with spectra characteristic of muconic semialdehydes can be made to accumulate in the media. These spectra indicate that benzoate is metabolised through catechol and 2-hydroxymuconic semialdehyde (2-hydroxy-6-oxohexa-2,4-dienoate), m-toluate through 3-methylcatechol and 2-hydroxy-6-oxohepta-2,4-dienoate and p-toluate through 4-methylcatechol and 2-hydroxy-5-methylmuconic semialdehyde (2-hydroxy-5-methyl-6-oxohexa-2,4-dienoate).2. Freshly harvested cells grown on all three substrates only consume oxygen a t a significant rate when presented with the growth substrates themselves and catechol and the methylcatechols, but not with salicylate, protocatechuate or phenol or the cresols.3. Extracts of cells grown on these substrates contain high induced levels of the suite of meta cleavage enzymes, including both the hydrolytic branch and the 4-oxalocrotonate branch.4. The substrate specificity of the early enzymes of the pathway suggests that only one nonspecific enzyme expresses each activity in cell-free extracts and that it is nonspecifically induced during growth on all three carbon sources. 5.It is suggested that the metabolic role of the hydrolytic branch of the pathway is the assimilation of 3-methylcatechol and its precursor, and that of the 4-oxalocrotonate branch is to assimilate catechol and 4-methylcatechol and their precursors.6. o-Toluate is not metabolised because it is neither a substrate for the benzoate oxidase system nor an inducer of any of the meta pathway enzymes.7 . It appears that benzoate and m-and p-toluates are the substrate inducers of the meta pathway enzymes in this organism. The ortho pathway is induced on incubating cells with catechol and the first inducer appears to be &,cis-muconate or possibly catechol itself.The coexistence of two enzymes able to degrade the meta cleavage product of catechol, 2-hydroxymuconic semialdehyde, was first demonstrated in cell-free extracts of both benzoate-grown Azotobacter species [l] and a naphthalene-grown Pseudomonas strain NCIB 9816 [2]. Sala-Trepat and Evans [3] elucidated the two resulting pathways, which after diverging a t 2-hydroxymuconic semialdehyde converge again a t 2-oxopent-4-enoate ; they concluded that in Azotobacter only the 4-oxalocrotonate branch, involving as the first step the NADf-dependent dehydrogenation of 2-hydroxymuconic semialdehyde, was of any metabolic significance since the 2-hydroxymuconic semialdehyde hydrolase was present Enzymes. Catechol 1,2-oxygenase or catechol: oxygen 1,2-oxidoreductase (EC 1.13.1.1); catechol 2,3-oxygenase or catecho1:oxygen 2,3-oxidoredoctase (EC 1.13.1.2); NAD nucleosidase or NAD glycohydrolase (EC 3.2.2.6). 21 Eur. J. Blochem., Vol.28 in only very low uninducible levels. I n NCIB 9816 all the enzymes of the 4-oxalocrotonate branch are present [4] and again it has been proposed that catechol is metab...

1.A reinvestigation of the catabolic pathway(s) used by Pseudomonas putida NCIB 10015 (Dagley's strain) for the degradation of phenol and the cresols has proved the existence of a metabolic divergence after meta cleavage of the eatechols formed by hydroxylation of the primary substrates. The ring-fission products of catechol and 4-methylcatechol are shown to be simultaneously catabolized by two different enzymic activities, an NADf-dependent dehydrogenase and a cofactor-independent hydrolase. The metabolizing activitics of bot,h ring-fission products in extracts of cells grown on phenol and the cresols (0-, m-and p-cresol) were found to be nonspecific ; thermal inactivation of extracts of phenol-grown cells has shown that this nonspecificity is attributable to only one enzyme expressing each activity and that the two activities are locatcd on separate proteins.2. Extracts of cells grown on all four substrates contain high induced levels of the meta cleavage suite of enzymes functional in the dissimilation of catechol, including both the 4-oxalocrotonate branch (NADf-dependent 2-hydroxymuconic semialdehyde dehydrogenase, 4-oxalocrotonate tautomerase and 4-oxalocrotonate decarboxylase) and the hydrolytic branch (2-hydroxymuconic semialdehyde hydrolase).3. The hydroxylase, oxygenase, dehydrogenase and hydrolase activities are shown to be nonspecific and can also act upon the methyl derivatives of their respective substrates. A constant pattern of specificity was found for these enzymes, independent of the monophenolic substrate used for growth.4. From studies with a mutant lacking phenol hydroxylase, the entire suite of meta cleavage enzymes are shown to be coincidently induced from the top by the primary substrate (phenol or the cresols) .5. The evolutionary and physiological implications of the divergent pathway are discussed. [2,3], and that involving the 2-hydroxymuconic semialdehyde dehydrogenase (the 4-oxalocrotonate branch) bears similarities to that