Lolita M. Corpuz

Sequence of a cDNA and expression of the gene encoding epidermal and gut chitinases of Manduca sexta

Insects use chitinolytic enzymes to digest chitin in the exoskeleton during the molting process. We have isolated and sequenced a chitinase-encoding cDNA from the tobacco hornworm, Manduca sexta, compared its sequence with genes encoding chitinolytic enzymes from other sources, and studied chitinase gene expression and hormonal regulation during the larval-pupal transformation. The insert DNA in this clone is 2452 nucleotides long with an open reading frame of 1662 nucleotides that encodes a protein of 554 amino acids with a molecular weight of 62 kDa. Several regions of the amino acid sequence in this protein are similar to sequences in yeast, cucumber and bacterial endo-beta-N-acetylglucosaminidases. Hybrid-selection of mRNA and in vitro translation yielded an immunoreactive protein with an apparent molecular mass of 75 kDa, which is similar to the size of a chitinase present in pharate pupal molting fluid. Southern blot analysis indicated that one or two genes related to the cDNA clone are encoding chitinases in the Manduca genome. The major tissues expressing chitinase genes were the epidermis and gut with mRNA levels highest on c. days 5-7 during the fifth larval instar. Injection of 20-hydroxyecdysone into ligated fifth instar abdomens caused about a 10-fold increase in mRNA levels in both epidermis and gut, and topical application of the juvenile hormone mimic, fenoxycarb, suppressed the ecdysteroid-induced accumulation of chitinase RNA.

Mimecan, the 25-kDa Corneal Keratan Sulfate Proteoglycan, Is a Product of the Gene Producing Osteoglycin

Bovine cornea contains three unique keratan sulfate proteoglycans (KSPGs), of which two (lumican and keratocan) have been characterized using molecular cloning. The gene for the third protein (KSPG25) has not been identified. This study examined the relationship between the KSPG25 protein and the gene for osteoglycin, a 12-kDa bone glycoprotein. The N-terminal amino acid sequence of KSPG25 occurs in osteoglycin cDNA cloned from bovine cornea. The osteoglycin amino acid sequence makes up the C-terminal 47% of the deduced sequence of the KSPG25 protein. Antibodies to osteoglycin reacted with intact corneal KSPG, with KSPG25 protein, and with a 36-kDa protein, distinct from lumican and keratocan. KSPG25-related proteins, not modified with keratan sulfate, were also detected in several connective tissues. Northern blot analysis showed mRNA transcripts of 2.4, 2.5, and 2.6 kilobases in numerous tissues with the 2.4-kilobase transcript enriched in ocular tissues. Ribonuclease protection analysis detected several protected KSPG25 mRNA fragments, suggesting alternate splicing of KSPG25 transcripts. We conclude that the full-length translation product of the gene producing osteoglycin is a corneal keratan sulfate proteoglycan, also present in many non-corneal tissues without keratan sulfate chains. The multiple size protein products of this gene appear to result from in situ proteolytic processing and/or alternative splicing of mRNA. The name mimecan is proposed for this gene and its products.

Integrated genomic approaches implicate osteoglycin (Ogn) in the regulation of left ventricular mass

Left ventricular mass (LVM) and cardiac gene expression are complex traits regulated by factors both intrinsic and extrinsic to the heart. To dissect the major determinants of LVM, we combined expression quantitative trait locus and quantitative trait transcript (QTT) analyses of the cardiac transcriptome in the rat. Using these methods and in vitro functional assays, we identified osteoglycin (Ogn) as a major candidate regulator of rat LVM, with increased Ogn protein expression associated with elevated LVM. We also applied genome-wide QTT analysis to the human heart and observed that, out of ∼22,000 transcripts, OGN transcript abundance had the highest correlation with LVM. We further confirmed a role for Ogn in the in vivo regulation of LVM in Ogn knockout mice. Taken together, these data implicate Ogn as a key regulator of LVM in rats, mice and humans, and suggest that Ogn modifies the hypertrophic response to extrinsic factors such as hypertension and aortic stenosis.

Sequences of two cDNAs and expression of the genes encoding methionine-rich storage proteins of Manduca sexta☆

Abstract In Manduca sexta, storage proteins accumulate during the final larval stadium for utilization during subsequent larval-pupal-adult transformations. Two cDNA clones (designated clone 119 and clone 201), that represent two distinct but related genes (42% sequence identity), were isolated from a cDNA library prepared from day 7 fifth instar larval fat body and found to encode two different storage proteins synthesized during the last larval instar. Northern blot analyses revealed that the two clones hybridize to 2.4 kb transcripts that are translated to 79 kDa protein products during in vitro translation experiments. Clone 119 encodes a methionine-rich storage protein, designated as SP1A, that shares 37% sequence identity with the Bombyx mori sex-specific storage protein SP1. Clone 201, on the other hand encodes a storage protein, designated as SP1B, that is more closely related to B. mori SP1 (63% identity), and is probably identical to the Manduca female-specific storage protein (FSP). Insert DNA from clone 201, but not clone 119, cross-hybridizes to that of FSP cDNA (Webb and Riddiford, Dev. Biol.130, 671–691, 1988a). Both storage proteins are synthesized only in the fat body and only during the fifth larval stadium, indicating tissue- and stage-specific expression of the two genes. Both genes exhibit sex-specific differences in expression. In the fifth larval stadium, the mRNAs for the SP1A and SP1B proteins begin to accumulate at about day 2 in the female fat body but appear 2 or 3 days later in the male fat body. In both sexes SP1A mRNA remains relatively high beyond the time when SP1B mRNA has already declined to low levels, suggesting differences in mRNA stability or expression. Injection of 20-hydroxyecdysone into ligated fifth instar abdomens causes substantial increases in the levels of both mRNAs, whereas topical application of the juvenile hormone mimc, fenoxycarb, to feeding fifth instar larvae produces substantial declines in the mRNA levels, indicating hormonal effects at the transcriptional level. The data support the hypothesis that the expression of these M. sexta methionine-rich storage protein genes is stimulated by ecdysteroid and inhibited by juvenile hormone.

Differential splicing and alternative polyadenylation generate multiple mimecan mRNA transcripts.

We previously showed the 25-kDa corneal keratan sulfate proteoglycan to be a translation product of the gene producing osteoglycin and proposed the name mimecan for this gene and its product. We also demonstrated three mimecan RNA transcripts using Northern blot analysis. In this report, we investigate the mechanisms accounting for these transcripts. Ribonuclease protection analysis and reverse transcription-polymerase chain reaction of bovine corneal mRNA detected a mimecan transcript that lacked 278 base pairs of the 5′-untranslated region between residues 62 and 340. This splice variant represents the predominant form of mimecan mRNA in bovine cornea and sclera. It was also detectable in other bovine tissues as a minor transcript. Two additional cDNA clones that were isolated contained 398 bases of nucleotide sequence at the 3′-end of mimecan cDNA, not present in the published sequence. Ribonuclease protection analyses with the 3′-probe, which included the new sequence, allow detection of three RNA transcripts while 5′-probes recognized only two. These results indicate that the three canonical polyadenylation sites in the 3′-untranslated region of mimican mRNA are alternatively selected. Possible roles for this previously undetected degree of diversity of mimecan RNA isoforms transcribed in the same tissue are discussed.

Structure and Sequence of the Gene Encoding Human Keratocan

Keratocan is one of the three major keratan sulfate proteoglycans characteristically expressed in cornea. We have isolated cDNA and genomic clones and determined the sequence of the entire human keratocan (Kera) gene. The gene is spread over 7.65 kb of DNA and contains three exons. An open reading frame starting at the beginning of the second exon encodes a protein of 352 aa. The amino acid sequence of keratocan shows high identity among mammalian species. This evolutionary conservation between the keratocan proteins as well as the restricted expression of Kera gene in cornea suggests that this molecule might be important in developing and maintaining corneal transparency.

Molecular cloning and relative tissue expression of keratocan and mimecan in embryonic quail cornea.

We have cloned and sequenced the cDNAs for quail cornea keratan sulfate proteoglycan core proteins, keratocan and mimecan. The deduced quail keratocan protein contains a single conservative amino acid difference from the chick sequence, whereas quail mimecan protein contains a 58 amino acid-long avian-unique sequence that shares no homology with mammalian mimecan. Ribonuclease protection assay of Day 16 embryonic quail tissues reveals that keratocan and lumican are expressed at highest levels in cornea, whereas mimecan mRNA is expressed at a much lower level. Keratocan is expressed only in quail cornea, whereas mimecan is expressed in many different tissues as four transcripts of different sizes. Both lumican and mimecan are expressed at lowest levels in brain, liver and sternum.