Aspergillus niger produces several polygalacturonases that, with other enzymes, are involved in the degradation of pectin. One of the two previously characterized genes coding for the abundant polygalacturonases T and I1 (PGI and PGII) found in a commercial pectinase preparation was used as a probe to isolate five more genes by screening a genomic DNA library in phage lEMBL4 using conditions of moderate stringency. The products of these genes were detected in the culture medium of Aspergilh nidulans transformants on the basis of activity measurements and Western-blot analysis using a polyclonal antibody raised against PGI. These transformants were, with one exception, constructed using phage DNA. A . niduluns transformants secreted high amounts of PGI and PGII in comparison to the previously characterized A. niger transformants and a novel polygalacturonase (PGC) was produced at high levels by A. nidulans transformed with the subcloned pguC gene. This gene was sequenced and the protein-coding region was found to be interrupted by three introns; the different intronlexon organization of the three sequenced A. niger polygalacturonase genes can be explained by the gain or loss of two single introns. The pgaC gene encodes a putative 383-amino-acid prepro-protein that is cleaved after a pair of basic amino acids and shows approximately 60% amino acid sequence similarity to the other polygalacturonases in the mature protein. The N-terminal amino acid sequences of the A. niger polygalacturonases display characteristic amino acid insertions or deletions that are also observed in polygalacturonases of phytopathogenic fungi. In the upstream regions of the A . niger polygalacturonase genes, a sequence of ten conserved nucleotides comprising a CCAAT sequence was found, which is likely to rcpresent a binding site for a regulatory protein as it shows a high similarity to the yeast CYCl upstream activation site recognized by the HAP2/3/4 activation complex.Polygalacturonases are involved in the degradation orpectin, a complex polysaccharide that is primarily found in the middle lamella and primary cell wall of higher plants [I]. Polygalacturonases as well as other pectolytic enzymes are produced by many organisms (reviewed in [2]), and microbial pectolytic enzymes have been extensively studied in relation to plant pathogenesis. The molecular biology of the bacterial enzymes, especially the pectate lyases of Erwiniu, is well developed compared to that of the pectolytic enzymes of fungal pathogens [3, 41. Recently, genomic or cDNA clones coding for pectinesterase [5], pectin lyases [6, 71, pectate lyase [8] and polygalacturonases [9 -121 have been obtained from Aspei-gillus species, which are considered to be saprophytes. The availability of commercial pectinase preparations derived from Aspergillus niger has contributed to the rapid progress in gene Correspondence to J. Visser, Section

The structure and expression of the polygalacturonase-encoding pgaII genes of two recently recognized species, Aspergillus niger and Aspergillus tubigensis, was investigated. While the structure of the pgaII genes is very similar, showing 83% DNA sequence identity and 94% identity at the amino acid level, they have diverged significantly. The NH2-terminal sequence suggests that these PGs are made as pre pro-proteins and the secretory propeptide of the PGII precursors shows sequence homology with some other fungal pro-peptides. The expression of the pgaII genes is strongly regulated by the carbon source and the A. tubigensis gene is expressed and regulated in A. niger transformants. The low similarity of the fungal PGs with those of bacterial and plant origin is discussed in relation to the possible functional role of specific amino acids.

Polygalact~ron~eIlof ~spergjtZ~ niger was fragmented using CNBr and the NHr-terminal fragment and another fragment were partially sequenced. The poiygalacturonasell @gofI) gene was then isolated by using an oligonucleotide mixture based on the internal ammo acid sequence as a probe. The nucleotide sequence of thepgaI1 structural gene was determined. It was found that polygalacturonaseI1 is synthesized as a precursor having an NH,-terminal prepro-sequence of 27 amino acids. The cloned gene was used to construct polygalacturonaseI1 over-producing A. niger strains. PolygalacturonaseII was isolated from one such strain and was determined to be correctiy processed and to be fully active.

The filamentous fungus Aspergillus niger produces several endopolygalacturonases that are involved in the degradation of pectin. PGI, the enzyme representing the second most abundant activity in a commercial enzyme preparation, was further characterized and the corresponding gene was isolated. The nucleotide sequence of the pgaI gene was determined and the protein coding region was found to be interrupted by two short introns, one of which has a unusual donor splice site. The deduced 368 amino acids long protein with a putative prepropeptide of 31 amino acids shows 60% sequence identity to PGII in the mature protein. PGI overproducing A. niger strains were obtained by cotransformation with the cloned gene.

In vitro translation products of total RNA isolated from soybean nodules at successive stages of nodule development were analyzed by two-dimensional gel electrophoresis. In that way the occurrence of over 20 mRNAs specifically transcribed from nodulin genes was detected. The nodulin genes could be divided into two classes according to the time of expression during nodule development. Class A comprises at least 4 nodulin mRNAs which are found when a globular meristem is present in the root cortex. These class A nodulin genes have a transient expression. Class B nodulin genes are expressed when the formation of a nodule structure has been completed. Bradyrhizobium japonicum nod (+) fix(-)mutants, with large deletions spanning the nif H,DK region, still induced nodules showing normal expression of all nodulin genes, indicating that the nif H,DK region is not involved in the induction of nodulin genes. In nodules induced by Bradyrhizobium japonicum nod (+) fix(-)mutant HS124 the bacteria are rarely released from the infection thread and the few infected cells appear to be collapsed. All class A and class B nodulin genes are expressed in HS124 nodules with the exception of 5 class B genes.