Biosynthesis of the activated sulfate donor, adenosine 3'-phosphate 5'-phosphosulfate, involves the sequential action of two enzyme activities: ATP sulfurylase, which catalyzes the formation of adenosine 5'-phosphosulfate (APS) from ATP and free sulfate, and APS kinase, which subsequently phosphorylates APS to produce adenosine 3'-phosphate 5'-phosphosulfate. Oligonucleotide primers were derived from a human infant brain-expressed sequence tag putatively encoding a portion of APS kinase. Using these primers, reverse transcriptase-polymerase chain reaction was performed on mRNA from neonatal normal mice resulting in amplification of a 127-bp DNA fragment. This fragment was subsequently used to screen a mouse brain lambda gt11 cDNA library, yielding a 2.2-kb clone. Primers were designed from the 5'-end of the 2.2-kb clone, and 5'-rapid amplification of cDNA ends was used to obtain the translation start site. Sequence from the overlapping clones was assembled into a 2475-bp composite sequence, which contains a single open reading frame that translates into a 624-deduced amino acid sequence. Northern blots of total RNA from neonatal mice yielded a single message species at approximately 3.3 kb. Southern blot of genomic DNA digested with several restriction enzymes suggested the gene is present as a single copy. Comparison against sequence data bases suggested the composite sequence was a fused sulfurylase-kinase product, since the deduced amino acid sequence showed extensive homology to known separate sequences of both ATP sulfurylase and APS kinase from several sources. The first 199 amino acids corresponded to APS kinase sequence, followed by 37 distinct amino acids, which did not match any known sequence, followed by 388 amino acids that are highly homologous to known ATP sulfurylase sequences. Finally, recombinant enzyme expressed in COS-1 cells exhibited both ATP sulfurylase and APS kinase activity.

Aggrecan is a large chondroitin sulfate proteoglycan, the expression of which is both tissue-specific and developmentally regulated. Here we report the cloning and sequencing of the 1.8-kilobase genomic 5' flanking sequence of the chick aggrecan gene and provide a functional and structural characterization of its promoter and enhancer region. Sequence analysis reveals potential Sp1, AP2, and NF-I related sites, as well as several putative transcription factor binding sites, including the cartilage-associated silencers CIIS1 and CIIS2. A number of these transcription factor binding motifs are embedded in a sequence flanked by prominent inverted repeats. Although lacking a classic TATA box, there are two instances in the 1.8-kb genomic fragment of TATA-like TCTAA sequences, as have been defined previously in other promoter regions. Primer extension and S1 protection analyses reveal three major transcription start sites, also located between the inverted repeats. Transient transfections of chick sternal chondrocytes and fibroblasts with reporter plasmids bearing progressively reduced portions of the aggrecan promoter region allowed mapping of chondrocyte-specific transcription enhancer and silencer elements that are consistent with the sequence analysis. These findings suggest the importance of this regulatory region in the tissue-specific expression of the chick aggrecan gene.

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