A Novel Set of Hepatic mRNAs Preferentially Expressed during an Acute Inflammation in Rat Represents Mostly Intracellular Proteins

A cloning of hepatic cDNAs associated with the early phase of an acute, systemic inflammation was carried out by differential screening of arrayed cDNA clones from rat livers obtained at 4-8 h postchallenge with Freund's complete adjuvant. End sequencing of 174 selected clones provided three cDNA groups that coded for: (i) 23 known acute-phase proteins, (ii) 31 known proteins whose change in hepatic synthesis during an acute phase was so far unsuspected, and (iii) 36 novel proteins whose cDNAs were completely sequenced. For 16 proteins in the third group the hepatic mRNA could be detected and quantitated by Northern blot hybridization in Freund's adjuvant-challenged animals, and an extrahepatic expression in healthy animals was further investigated. Matching the open reading frames of the 36 novel proteins with general and specialized data libraries indicated the potential relationships of 16 of these proteins with known protein families/superfamilies and/or the presence of functional domains previously described in other proteins. Overall, our search for novel inflammation-associated proteins selected mostly known or as yet undescribed proteins with an intracellular or membrane location, which extends our knowledge of the proteins involved in the intracellular metabolism of hepatic cells during a systemic, acute-phase response. Finally, some of the cDNAs above allowed us to successfully identify hepatic mRNAs that are differentially expressed in acute vs chronic (polyarthritis) inflammatory conditions in rat.     

Two cDNAs encoding novel human FGF receptor.

Two types of cDNAs encoding novel human FGF receptors were isolated. These two cDNAs were found to be closely related to the oncogene bek. Products from these genes were membrane-bound when their cDNAs were transiently expressed in COS cells, whereas products from the regions coding extracellular domains were free of membrane attachment and found in the culture medium.     

The alpha 1 chain of type XIII collagen consists of three collagenous and four noncollagenous domains, and its primary transcript undergoes complex alternative splicing.

We have recently reported a characterization of cDNA clones that encode an apparently novel human collagen that undergoes alternative splicing. These cDNAs covered one-third of the corresponding 2.5-2.8-kilobase mRNAs. We have now determined the complete primary structure of the protein encoded by several overlapping cDNAs isolated from a human endothelial cell library. Since the deduced translation product of the cDNAs is different in structure from all other collagen types, we have given the collagen chain encoded by the cDNAs the designation alpha 1 (XIII). The deduced polypeptide consists of three collagenous domains and four noncollagenous domains, two of them separating the collagenous domains and two located at the N-terminal and C-terminal ends of the polypeptide. Cysteine residues are found in three of the noncollagenous domains and also in the extreme N-terminal collagenous domain. Surprisingly, comparison of the nucleotide sequences encoded by the overlapping cDNA clones demonstrates that there are several alpha 1 (XIII) collagen mRNAs in HT-1080 human fibrosarcoma cells and human endothelial cells which differ in coding potential. Nuclease S1 mapping experiments suggest that these different mRNAs arise through alternative splicing of the precursor RNA at five locations within the coding region. This property makes type XIII collagen unique among all the collagen types studied so far. Its polypeptide length, therefore, may vary between 614 and 526 amino acids, depending on what internal splicing has taken place.     

Identification of membrane-bound and secreted proteins from Echinococcus granulosus by signal sequence trap

The signal sequence trap technique was applied to identify genes coding for secreted and membrane bound proteins from Echinococcus granulosus, the etiologic agent of cystic hydatid disease. An E. granulosus protoscolex cDNA library was constructed in the AP-PST vector such that randomly primed cDNAs were fused with a placental alkaline phosphatase reporter gene lacking its endogenous signal peptide. E. granulosus cDNAs encoding a functional signal peptide were selected by their ability to rescue secretion of alkaline phosphatase by COS-7 cells that had been transfected with the cDNA library. Eighteen positive clones were identified and sequenced. Their deduced amino acid sequences showed significant similarity with amino acid transporters, Krebs cycle intermediates transporters, presenilins and vacuolar protein sorter proteins. Other cDNAs encoded secreted proteins without homologues. Three sequences were transcribed antisense to E. granulosus expressed sequence tags. All the mRNAs were expressed in protoscoleces and adult worms, but some of them were not found in oncospheres. The putative E. granulosus secreted and membrane bound proteins identified are likely to play important roles in the metabolism, development and survival in the host and represent potential targets for diagnosis, drugs and vaccines against E. granulosus.     

Expressed sequence tags of Chinese cabbage flower bud cDNA.

We randomly selected and partially sequenced cDNA clones from a library of Chinese cabbage (Brassica campestris L. ssp. pekinensis) flower bud cDNAs. Out of 1216 expressed sequence tags (ESTs), 904 cDNA clones were unique or nonredundant. Five hundred eighty-eight clones (48.4%) had sequence homology to functionally defined genes at the peptide level. Only 5 clones encoded known flower-specific proteins. Among the cDNAs with no similarity to known protein sequences (628), 184 clones had significant similarity to nucleotide sequences registered in the databases. Among these 184 clones, 142 exhibited similarities at the nucleotide level only with plant ESTs. Also, sequence similarities were evident between these 142 ESTs and their matching ESTs when compared using the deduced amino acid sequences. Therefore, it is possible that the anonymous ESTs encode plant-specific ubiquitous proteins. Our extensive EST analysis of genes expressed in floral organs not only contributes to the understanding of the dynamics of genome expression patterns in floral organs but also adds data to the repertoire of all genomic genes.     

Prediction of the coding sequences of mouse homologues of KIAA gene: II. The complete nucleotide sequences of 400 mouse KIAA-homologous cDNAs identified by screening of terminal sequences of cDNA clones randomly sampled from size-fractionated libraries.

We have accumulated information of the coding sequences of uncharacterized human genes, which are known as KIAA genes, and the number of these genes exceeds 2000 at present. As an extension of this sequencing project, we recently have begun to accumulate mouse KIAA-homologous cDNAs, because it would be useful to prepare a set of human and mouse homologous cDNA pairs for further functional analysis of the KIAA genes. We herein present the entire sequences of 400 mouse KIAA cDNA clones and 4 novel cDNA clones which were incidentally identified during this project. Most of clones entirely sequenced in this study were selected by computer-assisted analysis of terminal sequences of the cDNAs. The average size of the 404 cDNA sequences reached 5.3 kb and that of the deduced amino acid sequences from these cDNAs was 868 amino acid residues. The results of sequence analyses of these clones showed that single mouse KIAA cDNAs bridged two different human KIAA cDNAs in some cases, which indicated that these two human KIAA cDNAs were derived from single genes although they had been supposed to originate from different genes. Furthermore, we successfully mapped all the mouse KIAA cDNAs along the genome using a recently published mouse genome draft sequence.