Molecular cloning and expression of two chicken invariant chain isoforms produced by alternative splicing

The biosynthesis of distinct forms of the invariant chain (Ii) protein from a unique gene as the result of differential splicing patterns has been observed in humans and mice. However, there have been no reports on the existence of Ii isoforms in avian species. In the present study, we identified two chicken Ii cDNAs by RT-PCR and RACE, and examined the Ii gene copy number, mRNA expression and protein expression by Southern blotting, Northern blotting and immunofluorescence confocal microscopy, respectively. One of the Ii cDNAs, named Ii-1, was 1,151 bp in length, and had an open reading frame (ORF) of 672 nucleotides, in agreement with a previously identified chicken Ii sequence; the other, named Ii-2, was 1,337 bp long and had an ORF of 861 nucleotides. Southern blotting confirmed that these cDNAs were derived from a single copy gene. Northern blotting performed with total RNA from various tissues of 6-week-old chickens revealed high levels of Ii-1 and Ii-2 mRNA expression in the spleen and bursa of Fabricius, and low levels of Ii-1 expression in the thymus, heart and liver, while Ii-2 was not expressed in these tissues. High levels of expression of both Ii isoforms were detected in the spleen and bursa of Fabricius during late embryogenesis. Immunofluorescence staining showed that Ii proteins were expressed in the cell membranes of the splenocytes. These data suggest that chicken Ii exists in two isoforms resulting from alternative splicing, and is strongly expressed in the major immune organs.     

Developmental expression of two Haliotis asinina hemocyanin isoforms

Hemocyanins are large copper-containing respiratory proteins that play a role in oxygen transport in many molluscs. In some species only one hemocyanin isoform is present while in others two are expressed. The physiological relevance of these isoforms is unclear and the developmental and tissue-specific expression of hemocyanin genes is largely unknown. Here we show that two hemocyanin genes in the gastropod Haliotis asinina, which encode H. asinina hemocyanin (HaH1) and HaH2 isoforms, are developmentally expressed. These genes initially are expressed in a small number of mesenchyme cells at trochophore and pre-torsional veliger stages, with HaH1 expression slightly preceding HaH2. These cells largely are localized to the visceral mass, although a small number of cells are present in head and foot regions. Following metamorphosis the isoforms show overlapping as well as isoform-specific expression profiles, suggesting some degree of isoform-specific function.     

Identification of two novel alternatively spliced Neuropilin-1 isoforms

Neuropilin-1 (NRP1) is a coreceptor to a tyrosine kinase receptor for both the vascular endothelial growth factor (VEGF) family and semaphorin (Sema) family members. NRP1 plays versatile roles in angiogenesis, axon guidance, cell survival, migration, and invasion. NRP1 contains three distinct extracellular domains, a1a2, b1b2, and c. We report here the identification of two novel soluble human NRP1 isoforms, which we named sIIINRP1 and sIVNRP1. These soluble NRP1 isoforms were generated by alternative splicing of the NRP1 gene, a common regulatory mechanism occurring in cell surface receptor families. Both sIIINRP1 and sIVNRP1 contain a1a2 and b1b2 domains, but no c domain, and the rest of the NRP1 sequence. Additionally, sIIINRP1 is missing 48 amino acids within the C-terminus of the b2 domain. Both sIIINRP1 and sIVNRP1 are expressed in human cancerous and normal tissues. These molecules are capable of binding to VEGF165 and Sema3A. Furthermore, recombinant sIIINRP1 and sIVNRP1 proteins inhibit NRP1-mediated MDA-MB-231 breast cancer cell migration. These results indicate the multiple levels of regulation in NRP1 function and suggest that these two novel NRP1 isoforms are useful antagonists for NRP1-mediated cellular activities.     

Primary structure and functional expression of h-caldesmon complementary DNA

Recently, the two Mr forms of caldesmon (Mr's in the range of 120-150kDa and 70-80kDa as judged by SDS-PAGE) have been identified. h-Caldesman (high Mr 120-150kDa caldesmon) is predominantly expressed in smooth muscles, and l-caldesmon (low Mr 70-80kDa caldesmon) in non-muscle cells. In this paper, we report the nucleotide sequence of chick embryo gizzard h-caldesmon cDNA and its translation into amino acid sequence. This sequence predicts a protein of 771 amino acids with a Mr of 88,743. The central portion of this sequence is composed of a 10-fold repeat of conserved amino acid sequence containing 13-15 amino acids. Further, a recombinant protein produced in Escherichia coli containing the full-length h-caldesmon cDNA has been characterized. Although the Mr of h-caldesmon predicted from amino acid sequence is 88,743, native and recombinant proteins show the same mol. wt. with 150kDa as measured by SDS-PAGE. This discrepancy may be due to the acidic amino acid-rich sequences at the N-terminal and central portions. A recombinant protein produced in E. coli possesses calmodulin-, F-actin- and tropomyosin-binding abilities in common with the native h-caldesmon.     

Receptor-mediated chicken oocyte growth: differential expression of endophilin isoforms in developing follicles

Receptor-mediated endocytosis of yolk precursors via clathrin-coated structures is the key mechanism underlying rapid chicken oocyte growth. In defining oocyte-specific components of clathrin-mediated events, we have to date identified oocyte-specific yolk transport receptors, but little is known about the oocytes' supporting endocytic machinery. Important proteins implicated in clathrin-mediated endocytosis and recycling are the endophilins, which thus far have been studied primarily in synaptic vesicle formation; in the present study, as a different highly active endocytic system, we exploit rapidly growing chicken oocytes. Molecular characterization of the chicken endophilins I, II, and III revealed that their mammalian counterparts have been highly conserved. All chicken endophilins interact via their SH3 domain with the avian dynamin and synaptojanin homologues and, thus, share key functional properties of mammalian endophilins. The genes show different expression patterns: As in mammals, expression is low to undetectable in the liver and high in the brain; in ovarian follicles harboring oocytes that are rapidly growing via receptor-mediated endocytosis, levels of endophilins II and III, but not of endophilin I, are high. Immunohistochemical analysis of follicles demonstrated that endophilin II is mainly present in the theca interna but that endophilin III predominates within the oocyte proper. Moreover, in a chicken strain with impaired oocyte growth and absence of egg-laying because of a genetic defect in the receptor for yolk endocytosis, endophilin III is diminished in oocytes, whereas endophilin III levels in the brain and endophilin II localization to theca cells are unaltered. Thus, the present study reveals that the endophilins differentially contribute to oocyte endocytosis and development.     

Cloning and sequence analysis of complementary DNA encoding a precursor for chicken natriuretic peptide

Chicken -natriuretic peptide (-chNP) has been identified in chicken heart, which showed higher homology to brain natriuretic peptide (BNP) than to atrial natriuretic peptide (ANP) [1]. Complementary DNA (cDNA) clone encoding a chNP precursor (pre-chNP) precursor (pre-chNP) was isolated from cardiac cDNA library and sequenced. Pre-chNP was 140-residue signal peptide at the N-terminus and -chNP at the C-terminus, and did not exhibit high homology to poreine BNP except for the C-terminal region. However, a characteristic AT-rich nucleotide sequence commonly found in mammalian BNPs was also present in the 3′-untranslated region. Thus, chNP is concluded to be classified into the BNP-type     

The expression of myosin heavy chain isoforms in normal and hypertrophied chicken slow muscle

Hypertrophy was produced in the anterior latissimus dorsi (ALD) muscle of 5-wk-old chickens by application of a load to the humerus. After 4 wk, hypertrophied ALD muscles were greater than 2.5 times heavier than contralateral control ALD muscles. Two isomyosins are distinguishable in normal ALD muscles by their different electrophoretic mobilities. It is shown here that the faster migrating SM-1 isomyosin decreases in abundance with age and that the application of an overload enhances both the rate and extent of this process. Monoclonal antibodies were selected by an immunotransfer technique that were specific for the heavy chains associated with either SM-1 or SM-2, or cross-reacted with both isoforms. The cellular distribution of the SM-1 and SM-2 isomyosins was analyzed by immunofluorescent technique using these antibodies. Anti-SM-1 and anti-SM-2 antibodies reacted with separate populations of cells, whereas the third antibody reacted with all myocytes in the normal ALD muscle. These data suggest that there is an exclusive cellular distribution of myosin heavy chains associated with SM-1 and SM-2 proteins. Immunofluorescent analysis of hypertrophied muscle showed the anti-SM-2-specific antibody reacting with all myocytes, whereas the anti-SM-1-specific antibody reacted with none. This is consistent with the elimination of the SM-1 isoform in hypertrophied muscles.     

Molecular cloning and characterization of two novel pro-apoptotic isoforms of caspase-10

Caspase-10/a (Mch4) and caspase-10/b (FLICE2) are related death effector domain-containing cysteine aspartases presumed to be at or near the apex of apoptotic signaling pathways. We report the cloning and characterization of two novel proteins that are splice isoforms of the caspase-10 family. Caspase-10/c is a truncated protein that is essentially a prodomain-only form of the caspase that lacks proteolytic activity in vitro but efficiently induces the formation of perinuclear filamentous structures and cell death in vivo. Caspase-10/c mRNA is specifically up-regulated upon TNF stimulation, suggesting a potential role of this isoform in amplifying the apoptotic response to extracellular stimuli such as cytokines. Caspase-10/d is a hybrid of the known caspases Mch4 and FLICE2, as it is identical to FLICE2 except for the small (p12) catalytic subunit, which is identical to Mch4. Caspase-10/d is proteolytically active in vitro and also induces cell death in vivo, although it is less active than Mch4. The mRNAs for all known isoforms of caspase-10 are abundantly expressed in fetal lung, kidney, and skeletal muscle but are very poorly expressed or absent in these tissues in the adult, implying a possible role for the caspase-10 family in fetal development.     

High-level expression of complementary DNA encoding rat calmodulin in Escherichia coli

We report the production and characterization of a rat calmodulin made in Escherichia coli. To express the rat calmodulin cDNA in E. coli, we have employed an expression vector containing the E. coli trp promoter and trpA terminator. The cDNA was modified so as to delete the 5' nontranslated sequence and to incorporate a consensus sequence for the E. coli ribosome-binding site. Several codons for the N-terminal amino acids were selected to fit the E. coli consensus nucleotide sequence around the translational initiation codon. After induction of expression in E. coli, rat calmodulin accounted for over 30% of total cellular proteins. About 100 mg of recombinant rat calmodulin, purified to over 90% homogeneity by extraction from bacterial lysate followed by phenyl-Sepharose column chromatography, was obtained from 1 liter of E. coli culture. This recombinant calmodulin activated rat brain cyclic AMP phosphodiesterase to the same extent as the native calmodulin purified from rat brain. These results indicate that the overproduction system of the recombinant calmodulin in E. coli facilitates the study of the structure-function relationship by site-specific mutagenesis.