Tissue-specific expression in the salivary glands of transgenic mice.

Using a DNA construct, named Lama, derived from the murine parotid secretory protein (PSP) gene, we have obtained salivary gland specific gene expression in transgenic mice. Lama is a PSP minigene and allows analysis of the PSP gene 5' regulatory region by transgenesis. We show here that the regulatory region included in Lama with 4.6 kb of 5' flanking sequence is sufficient to direct expression specifically to the salivary glands. The expression level in the parotid gland is only about one percent of the PSP mRNA level, while that of the sublingual gland is near the PSP mRNA level. This suggests significant differences in the PSP gene regulation in the two glands. In addition, Lama is a secretory expression vector in which cDNAs or genomic fragments can be inserted. We demonstrate that the Lama construct can direct the expression of a heterologous cDNA encoding the C-terminal peptide of human factor VIII to salivary glands and that the corresponding peptide is secreted into saliva.     

Molecular cloning of mouse PSP mRNA.

PSP is the most abundant translation product of mouse parotid glands where its production is co-ordinated with that of salivary amylase. The synthesis of these two proteins apparently is restricted to this tissue. In order to enable us to study common regulatory elements in the genes of the two proteins, double stranded cDNA, synthesized for parotid gland poly (A)+ RNA, was cloned. DNA sequencing of three clones complementary to the most abundant messenger indicated overlap and resulted in a total sequence of 867 nucleotides. Translation of this sequence revealed that at one end the amino acid sequence was the same as the N-terminal sequence of PSP. The sequence contains 60 nucleotides coding for part of or the complete signal peptide, 645 nucleotides coding for the PSP protein, and 162 nucleotides that apparently are not translated. Southern blot analysis suggests a simple structure for the PSP gene in mouse and man.     

Coordination of murine parotid secretory protein and salivary amylase expression.

PSP, parotid secretory protein, and salivary amylase are the major secretory proteins of mouse parotid gland where they appear in a constant ratio. Here we describe the isolation of the PSP gene and show through expression analysis on this and the salivary amylase gene that the two genes are transcribed in a coordinate fashion in adult animals, whereas the activation profiles are different during postnatal development. An explanation is put forward that involves activation of the genes at different stages of the acinar cell differentiation, leading in adults to the maximal and thus proportionate expression.     

Parotid secretory protein is an HDL-associated protein with anticandidal activity

High-density lipoprotein (HDL) is part of innate immunity, protecting against infection and inflammation. Using a proteomic approach, we identified an amino acid sequence in a hamster HDL protein that showed homology to rat and mouse parotid secretory protein (PSP), a salivary protein secreted from the parotid glands. We cloned the cDNA encoding a putative hamster homolog of rat and mouse PSP. Searches for conserved domains of the protein showed that the COOH terminus of hamster PSP contains a region homologous to the NH2 termini of a family of HDL-associated proteins, including LPS-binding protein, cholesteryl ester transfer protein, and phospholipid transfer protein. In mice, PSP was also associated with HDL but was not detected in very-low-density lipoprotein, low-density lipoprotein, or lipoprotein-deficient sera. In addition to salivary glands, we found that PSP mRNA was expressed in lung, testis, and ovary. The level of PSP in HDL was increased after endotoxin injection in hamsters, but not in mice. Recombinant PSP inhibits growth of Candida albicans in culture. In summary, our results showed that PSP is a novel anticandidal protein associated with HDL.     

Molecular cloning and nucleotide sequence of tuna growth hormone cDNA

cDNA for mRNA of tuna growth hormone (GH) was cloned by screening a cDNA library constructed from tuna pituitary gland poly(A)⁺ RNA. The nucleotide sequence of cDNA (911 bases) revealed an open reading frame of 615 nucleotides, including a sequence (51 bases) for a possible secretory protein leader peptide. Noncoding regions were found in the nucleotide sequences up- (5′-terminal: 65 bases) and down- (3′-terminal: 231 bases) stream of the open reading frame. An amino-acid sequence deduced from the nucleotide sequence of the cDNA was identical with that determined in the purified tuna GH. Tuna GH was composed of 187 amino acids, and had a calculated molecular weight of 21275. Amino-acid sequencing showed that there was one possible N-glycosylation site at Asn (Asn-Cys-Thr). Tuna GH showed amino-acid sequence homologies with chum salmon (67%), yellow tail (90%) and with human (32%) growth hormones.     

Cloning and complete nucleotide sequence of mouse immunoglobulin gamma 1 chain gene.

The 6.6 kb DNA fragment coding for the immunoglobulin γ1 chain was cloned from newborn mouse DNA using λgtWES·λB as the EK2 vector. The complete nucleotide sequence (1823 bases) of the γ1 chain gene was determined. The cloned gene contained the entire constant region gene sequence as well as the poly(A) addition site, but not the variable region gene. The results indicate that the variable and constant region genes of immunoglobulin heavy chain are separated in newborn mouse DNA. The constant region genes of other gamma chains (that is, γ2a, γ2b and γ3) are not present in the cloned DNA fragment. The sequence demonstrates that the γ1 chain gene is interrupted by three intervening sequences at the junction of the domains and the hinge region, as previously shown in the γ2b and α chain genes and in the γ1 chain gene cloned from myeloma. The results suggest that the intervening sequence was introduced into the heavy chain gene before divergence of the heavy chain classes, and also support the hypothesis that the splicing mechanism has facilitated the evolution of eucaryotic genes by linking duplicated domains or prototype peptides not directly adjacent to one another. Comparison of the nucleotide sequence of the γ1 chain gene around the boundaries of the coding and intervening sequences with those of other mouse genes revealed extensive divergence, although short prevalent sequences of AG-GTCAG at the 5′ border of the intervening sequence and TCTGCAG-GC at the 3′ border were deduced. A limited homology of nucleotide sequences was found among domains and between the hinge region and the 5′ portion of the CH2 domain. Comparison of 3′ untranslated sequences from the γ1 and γ2b chain genes and the mouse major β-globin gene shows significant homology and a palindrome sequence surrounding the poly(A) addition site.     

The BSP30 salivary proteins from cattle,LUNX/PLUNC and von Ebner's minor salivary gland protein are members of the PSP/LBP superfamily of proteins

Saliva influences rumen function in cattle, yet the biochemical role for most of the bovine salivary proteins (BSPs) has yet to be established. Two cDNAs (BSP30a and BSP30b) from bovine parotid salivary gland were cloned and sequenced, each coding for alternate forms of a prominent protein in bovine saliva. The BSP30 cDNAs share 96% sequence identity with each other at the DNA level and 83% at the amino acid level, and appear to arise from separate genes. The predicted BSP30a and BSP30b proteins share 26-36% amino acid identity with parotid secretory protein (PSP) from mouse, rat and human. BSP30 and PSP are in turn more distantly related to a wider group of proteins that includes lung-specific X protein, also known as palate, lung, and nasal epithelium clone (LUNX/PLUNC), von Ebner's minor salivary gland protein (VEMSGP), bactericidal permeability increasing protein (BPI), lipopolysaccharide binding protein (LBP), cholesteryl ester transfer protein (CETP), and the putative olfactory ligand-binding proteins RYA3 and RY2G5. Bovine cDNAs encoding homologs of LUNX/PLUNC and VEMSGP were isolated and sequenced. Northern blot analysis showed that LUNX/PLUNC, BSP30 and VEMSGP are expressed in bovine salivary tissue and airways, and that they have non-identical patterns of expression in these tissues. The expression of both BSP30a and BSP30b is restricted to salivary tissue, but within this tissue they have distinct patterns of expression. The proximity of the human genes coding for the PSP/LBP superfamily on HSA20q11.2, their similar amino acid sequence, and common exon segmentation strongly suggest that these genes evolved from a common ancestral gene. Furthermore, they imply that the BSP30a and BSP30b proteins may have a function in common with other members of this gene family.