Induction of four heat-shock proteins and their mRNAs in rat after whole-body hyperthermia

When the body temperature of rats was brought to 42 degrees C, four heat-shock proteins, with molecular weights of 70,000, 71,000, 85,000, and 100,000 (hsp 70, hsp 71, hsp 85, and hsp 100, respectively), were induced in various tissues of the rats. The hsp 70 was strongly induced by hyperthermia, and its accumulation was detected by Coomassie blue staining. The hsp 71 was abundant in various tissues of rats that were not heat-shocked. Analysis of translation products of liver mRNAs from heat-shocked rats also showed increased synthesis of the four heat-shock proteins, indicating that these hsp-mRNAs were induced after hyperthermia. Induction of the hsp-mRNAs was transient after hyperthermia. The four heat-shock proteins produced in various tissues after hyperthermia may be involved in homeostatic control in the heat-shock response of the rat.     

Molecular cloning of cDNA encoding human prointerleukin-6

The data are presented on the cloning and sequencing of cDNA coding for human interleukin-6. The variability of cDNA proIL-6 cloned from different cellular sources was studied. The variability of cDNA proIL-6 may be expressed as heterogeneity of 5'- and 3'-end sequences of cDNA as well as single base-pair changes.     

Molecular cloning of human satellite DNA sequences and their use in detecting DNA polymorphism

A approximately 400 bp HaeIII human genomic satellite DNA band was cloned into pUC18 to construct a partial library. A fragment of bacteriophage M13 containing a sequence homologous to the human minisatellite core was cloned in pUC18 and was used as a probe to isolate a approximately 350 bp human satellite clone (pTRF5.6) from the partial library. Other clones from this library showed a wide variation in terms of size and hybridization to the pTRF5.6 clone. Human DNA from different individuals was digested with restriction enzymes, Southern transferred and probed with TRF5.6. Individual-specific complex pattern of DNA bands was produced. TRF5.6, therefore, could be useful as a probe for detecting genetic polymorphism.     

Molecular cloning and expression in Escherichia coli of a cDNA encoding human pancreatic elastase 2

We have cloned a DNA that is complementary to the messenger RNA that encodes human pancreatic elastase 2 from a human pancreatic cDNA library using a cloned cDNA for rat pancreatic elastase 2 messenger RNA. This complementary DNA contains the entire protein coding region of 807 nucleotides which encodes preproelastase of 269 amino acids, and 4 and 82 nucleotides of the 5'- and 3'-untranslated sequences, respectively. When this deduced amino acid sequence was compared with known amino acid sequences it showed 82% homology with rat pancreatic elastase 2. This deduced sequence also contains a 16-amino-acid peptide identical with the N-terminal sequence determined for native human pancreatic proelastase 2. Taking the above findings together, we conclude that the cloned cDNA encodes a mature enzyme of 241 amino acids including 16 and 12 amino acids for a signal peptide and an activation peptide, respectively. Moreover, the predicted key amino acid residues involved in determining the substrate specificity of mammalian pancreatic elastase 2 are retained in the human enzyme. Cloned human pancreatic elastase 2 cDNA was expressed in E. coli as a mature and pro-form protein. Both resulting proteins showed immunoreactivity toward anti-elastase serum and enzymatic activity. We have also cloned and sequenced a porcine pancreatic elastase 2 cDNA.     

Small heat-shock proteins and their potential role in human disease

The elevated expression of stress proteins is considered to be a universal response to adverse conditions, representing a potential mechanism of cellular defense against disease and a potential target for novel therapeutics, including gene therapy and chaperone-modulating reagents. Recently, a single mutation in the small heat-shock protein human alphaB-crystallin was linked to desmin-related myopathy, which is characterized by abnormal intracellular aggregates of intermediate filaments in human muscle. New findings demonstrate that the high level of expression of stress proteins can contribute to an autoimmune response and can protect proteins that contribute to disease processes.     

Molecular cloning of the goose ACSL3 and ACSL5 coding domain sequences and their expression characteristics during goose fatty liver development

It has been demonstrated that ACSL3 and ACSL5 play important roles in fat metabolism. To investigate the primary functions of ACSL3 and ACSL5 and to evaluate their expression levels during goose fatty liver development, we cloned the ACSL3 and ACSL5 coding domain sequences (CDSs) of geese using RT-PCR and analyzed their expression characteristics under different conditions using qRT-PCR. The results showed that the goose ACSL3 (JX511975) and ACSL5 (JX511976) sequences have high similarities with the chicken sequences both at the nucleotide and amino acid levels. Both ACSL3 and ACSL5 have high expression levels in goose liver. The expression levels of ACSL3 and ACSL5 in goose liver and hepatocytes can be changed by overfeeding geese and by treatment with unsaturated fatty acids, respectively. Together, these results indicate that ACSL3 and ACSL5 play important roles during fatty liver development. The different expression characteristics of goose ACSL3 and ACSL5 suggest that these two genes may be responsible for specific functions.     

Molecular cloning and expression of a cDNA encoding the secretin receptor.

Secretin is a 27 amino acid peptide which stimulates the secretion of bicarbonate, enzymes and potassium ion from the pancreas. A complementary DNA encoding the rat secretin receptor was isolated from a CDM8 expression library of NG108-15 cell line. The secretin receptor expressed in COS cells could specifically bind the iodinated secretin with high and low affinities. Co-expression of the secretin receptor with the alpha-subunit of rat Gs protein increased the concentration of the high affinity receptor in the membrane fraction of the transfected COS cells. Secretin could stimulate accumulation of cAMP in COS cells expressing the cloned secretin receptor. The nucleotide sequence analysis of the cDNA has revealed that the secretin receptor consists of 449 amino acids with a calculated Mr of 48,696. The secretin receptor contains seven putative transmembrane segments, and belongs to a family of the G protein-coupled receptor. However, the amino acid sequence of the secretin receptor has no significant similarity with that of other G protein-coupled receptors. A 2.5 kb mRNA coding for the secretin receptor could be detected in NG108-15 cells, and rat heart, stomach and pancreatic tissue.     

Molecular cloning and sequence analysis of cDNA encoding human ferrochelatase

The cDNA encoding human ferrochelatase [EC 4.99.1.1] was isolated from a human placenta cDNA library in bacteriophage lambda gt11 by screening with a radiolabeled fragment of mouse ferrochelatase cDNA. The cDNA had an open reading frame of 1269 base pairs (bp) encoding a protein of 423 amino acid residues (Mr. 47,833) with alternative putative polyadenylation signals in the 3' non-coding regions and poly (A) tails. Amino acid sequencing showed that the mature protein consists of 369 amino acid residues (Mr. 42,158) with a putative leader sequence of 54 amino acid residues. The human enzyme showed an 88% identity to mouse enzyme and 46% to yeast enzyme. Northern blot analysis showed two mRNAs of about 2500 and 1600 bp for ferrochelatase in K562 and HepG2 cells. As full-length cDNA for human ferrochelatase is now available, molecular lesions related to erythropoietic protoporphyria can be characterized.     

Affinity isolation of heat-shock and other calmodulin-binding proteins following hyperthermia

The interaction of calmodulin (CaM) with heat-shock and other binding proteins was studied in rat adenocarcinoma cells. Cells were equilibrium-labeled for 48 h prior to heating for 1 h at 43 degrees C, or pulse-labeled for 2 h at 37 degrees C after heating, to monitor the effect of heat on the affinity of CaM-binding proteins synthesized under these conditions. A CaM antagonist shown to sensitize to heat killing, W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide], was used in competition assays to help monitor any changes in affinity. We found that heating tended to reduce the CaM-binding of proteins synthesized before heating relative to their 37 degrees C controls and proteins synthesized after heating tended to have increased binding relative to their respective controls. Members of the heat-shock protein (hsp) 90-, 70-, and 26-kDa families were among the proteins that bound to CaM and were eluted by W-7. The peak elution fractions for the hsp's and other cellular proteins varied, but hsp-70 eluted in the early fractions. The hsp-70 family was also found to be among a number of W-7-binding proteins. We conclude that the assumption that CaM antagonists potentiate killing of heated cells solely by competing nonspecifically for CaM-binding protein sites on CaM does not explain the process completely. These antagonists could also act by competing for CaM-binding sites with specific proteins whose interaction with CaM is important for survival following heating, or by directly binding to other proteins whose function is important for survival and inhibiting their activity. We do not have sufficient data to discern the predominant mechanism among these possibilities, but we believe all are likely to occur in heated cells and speculate that inhibition of the functions of the hsp-70 family is important in several of these antagonist actions.     

Molecular cloning and expression of the human interferon-gamma receptor

A cDNA encoding the human interferon-gamma receptor was isolated from a lambda gt11 expression library using a polyclonal antireceptor antiserum. The gene for this receptor was identified in a cosmid library and transfected into mouse cells. The human interferon-gamma receptor expressed in mouse cells displayed the same binding properties as in human cells. However, transfected cells were not sensitive to human IFN-gamma, suggesting the need for species-specific cofactors in receptor function. As inferred from the cDNA sequence, the human interferon-gamma receptor shows no similarities to known proteins and represents a novel transmembrane receptor. It is most likely the product of a single mRNA and a gene located on chromosome 6q.     

Molecular cloning and expression of human L-pipecolate oxidase

In higher eukaryotes L-lysine can be degraded via two distinct routes including the saccharopine pathway and the L-pipecolate pathway. The saccharopine pathway is the primary route of degradation of lysine in most tissues except the brain in which the L-pipecolate pathway is most active. L-pipecolate is formed from L-lysine via two enzymatic reactions and then undergoes dehydrogenation to Delta(1)-piperideine-6-carboxylate. At least in humans and monkeys, this is brought about by the enzyme L-pipecolate oxidase (PIPOX) localized in peroxisomes. In literature, several patients have been described with hyperpipecolic acidaemia. The underlying mechanism responsible for the impaired degradation of pipecolate has remained unclear through the years. In order to resolve this question, we have now cloned the human L-pipecolate oxidase cDNA which codes for a protein of 390 amino acids and contains an ADP-betaalphabeta-binding fold compatible with its identity as a flavoprotein. Furthermore, the deduced protein ends in -KAHL at its carboxy terminus which constitutes a typical Type I peroxisomal-targeting signal (PTS I).     

Molecular cloning and expression of the cDNA encoding feline granulocyte colony-stimulating factor.

Both genomic DNA and cDNA of the feline granulocyte colony-stimulating factor (G-CSF) gene were cloned from CRFK cells. Southern blot analysis showed that the haploid genome contains a single copy of the G-CSF gene. The RT-PCR analysis of several feline cell lines revealed expression of G-CSF mRNA in response to lipopolysaccharide stimulation. Sequence analysis of genomic and cDNA clones indicated that the intron-exon junction structure is conserved between the human and the feline G-CSF genes. The G-CSF coding region encodes a predicted protein of 195 amino acids including a signal sequence of 21 amino acids. The feline G-CSF amino acid sequence shares a high degree of identity with the canine (90.8%), human (87.4%), ovine (83.9%), bovine (82.8%), porcine (80.5%), murine (70.7%) and rat (66.8%) G-CSF. The feline G-CSF expressed in insect cells using recombinant baculovirus vector was biologically active as measured in a proliferation assay using NFS-60 cells and an induction assay of leukocytes in cats.