Characterization of a cDNA encoding a novel heat-shock protein that binds to calmodulin.

A cDNA clone (pTCB48) encoding a calmodulin-binding protein was isolated by screening a lambda ZAPII cDNA expression library constructed from cell cultures of heat-shocked tobacco (Nicotiana tabacum L. cv Wisconsin-38) with metabolically labeled [35S]calmodulin. Calmodulin gel overlay analysis indicated that pTCB48 generated major peptides of 53, 36, and 22 kD and two minor peptides of 37 and 16 kD that bound calmodulin in a Ca(2+)-dependent manner. Deletion analysis of pTCB48 indicated that these and the minor calmodulin-binding proteins resulted from the insert. A probe made from the cDNA insert recognized two bands with sizes of 2.1 and 1.8 kb on northern blot analysis. Both species of RNAs were undetectable in the control and were induced after 15 min of heat-shock treatment at 38 degrees C. The intensity of the two bands reached maximum after 1.5 h of heat-shock treatment. The cDNA clone was not full length; however, the complete sequence was determined by 5' rapid amplification of cDNA ends using nested antisense primers. The full-length cDNA contains 1648 bp and a single open reading frame of 1347 bp and is expected to encode a protein of approximately 50 kD. No significant homology with other reported genes and proteins was found. Structural predictions, deletion analysis, and gel overlay analysis suggested that the calmodulin-binding domain was a basic amphiphilic alpha-helix near the C terminus of the protein. The strong induction of the mRNA for this protein suggests a role for Ca2+/calmodulin-mediated process in the heat-shock response.     

Molecular cloning of cDNA encoding porcine interleukin-15

Interleukin-15 (IL-15) is a recently identified growth and differentiation factor with an important potential role in the initial immune responses to infection. To enable the study of the role of this cytokine in the protective immune-mechanisms generated against parasitic diseases of swine, cDNA was generated from a macrophage enriched adherent cell population from peripheral blood mononuclear cells (PBMC). This cDNA was used for the enzymatic amplification of the porcine IL-15 sequence using human IL-15-derived primers. The open-reading frame of the porcine IL-15 cDNA is 486 base pairs (bp) in length and encodes a 162-amino-acid (aa) protein. Comparisons of the predicted swine protein sequence with those predicted from human, bovine and mouse IL-15 sequences indicate similarities of 82.1, 84.6, and 71.6%, respectively.     

Molecular cloning and sequencing of a novel cDNA encoding for a protein involved in human sperm function.

A cDNA encoding for an antigen, designated as NZ-3, was cloned and sequenced from human testis. The 1481-bp NZ-3 cDNA yielded an open reading frame (ORF) of 231 amino acids (aa) with the first ATG, Met start codon at nucleotide (nt) 104 and the stop codon TGA at nt 797. Extensive computer search indicated it to be a novel cDNA/protein. The ORF of NZ-3 cDNA was subcloned into pGEX-1lambdaT vector and expressed in glutathione S-transferase gene fusion system. The expressed recombinant protein had a molecular size of approximately 25 kDa, and the rabbit antibodies (Ab) against the recombinant antigen recognized a specific protein band of 63 +/- 3 kDa in the human testis extract. The NZ-3 antigen was located on the acrosomal and tail regions of human sperm cell and the NZ-3 Ab significantly (P < 0.001) inhibited human sperm capacitation and/or acrosome reaction. The novel recombinant NZ-3 antigen may find applications in immunocontraception and in specific diagnosis of human infertility.     

Molecular cloning of an Arabidopsis cDNA encoding a dynamin-like protein that is localized to plastids

Dynamin-related proteins are high molecular weight GTPase proteins found in a variety of eukaryotic cells from yeast to human. They are involved in diverse biological processes that include endocytosis in animal cells and vacuolar protein sorting in yeast. We isolated a new gene, ADL2, that encodes a dynamin-like protein in Arabidopsis. The ADL2 cDNA is 2.68 kb in size and has an open reading frame for 809 amino acid residues with a calculated molecular mass of 90 kDa. Sequence analysis of ADL2 revealed a high degree of amino acid sequence similarity to other members of the dynamin superfamily. Among those members ADL2 was most closely related to Dnm1p of yeast and thus appears to be a member of the Vps1p subfamily. Expression studies showed that the ADL2 gene is widely expressed in various tissues with highest expression in flower tissues. In vivo targeting experiments showed that ADL2:smGFP fusion protein is localized to chloroplasts in soybean photoautroph cells. In addition experiments with deletion constructs revealed that the N-terminal 35 amino acid residues were sufficient to direct the smGFP into chloroplasts in tobacco protoplasts when expressed as a fusion protein.     

Characterization of a pollen-specific cDNA from sunflower encoding a zinc finger protein.

The maize transposable element Activator (Ac) carries subterminal CpG-rich sequences which are essential for the transposition of the element. It has previously been shown that the methylation of certain sequences contained in this region can alter their ability to interact with the Ac-encoded protein. The novel hypothesis that the methylation of subterminal Ac sequences is required for transposition was tested. Approximately 150 bp of the 5' subterminal region of the Ac element was examined for the presence of 5-methylcytosines by the ligation-mediated polymerase chain reaction (LMPCR)-aided genomic sequencing method. The methylation status of 22 and 39 cytosines on either strand of the DNA were analysed in each of five different transgenic tobacco cultures carrying transposable Ac sequences. Ten micrograms of tobacco DNA were used for each base-specific cleavage reaction before amplification by LMPCR. All but one of the cytosines were unmethylated. Only a minor fraction of the Ac molecules was methylated at one cytosine residue. It is concluded that DNA methylation at the tested Ac sequences is not required for the transposability of Ac or Ds elements in tobacco cells.     

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.     

Kin17, a mouse nuclear zinc finger protein that binds preferentially to curved DNA.

Kin17 is a 45 kDa protein encoded by the KIN17 gene located on mouse chromosome 2, band A. The kin17 amino acid sequence predicts two domains, which were shown to be functional: (i) a bipartite nuclear localization signal (NLS) that can drive the protein to the cell nucleus, (ii) a bona fide zinc finger of the C2H2 type. The zinc finger is involved in kin17 binding to double-stranded DNA since a mutant deleted of the zinc finger, kin17 delta 1, showed reduced binding. Single-stranded DNA was bound poorly by kin17. Interestingly, we found that kin17 protein showed preferential binding to curved DNA from either pBR322 or synthetic oligonucleotides. Binding of kin17 to a non-curved DNA segment increased after we had inserted into it a short curved synthetic oligonucleotide. Kin17 delta 2, a mutant deleted of 110 amino acids at the C-terminal end, still exhibited preferential binding to curved DNA and so did kin17 delta 1, suggesting that a domain recognizing curved DNA is located in the protein core.     

Molecular cloning of a novel ubiquitin-like protein, UBIN, that binds to ER targeting signal sequences

To identify proteins that interact with HSP47, an endoplasmic reticulum (ER)-resident molecular chaperone, a yeast two-hybrid screening was performed using mouse full-length HSP47 including an N-terminal signal sequence as a bait. Analysis of several positive clones led to the identification and cloning of a novel gene, ubin, encoding a ubiquitin-like protein. Unlike other ubiquitin-like proteins, UBIN was shown to interact with signal sequences of various secretory and ER-luminal proteins, including HSP47, but not interact with signal sequences of mitochondrial targeting in two-hybrid system. The possible function of UBIN will be discussed with regards to novel characteristics of binding to signal sequences for ER targeting.     

Inhibition of human telomerase activity by an engineered zinc finger protein that binds G-quadruplexes

The G-quadruplex nucleic acid structural motif is a target for designing molecules that could potentially modulate telomere length or have anticancer properties. We have recently described an engineered zinc finger protein (Gq1) that binds with specificity to the intramolecular G-quadruplex formed by the human telomeric sequence 5'-(GGTTAG)(5)-3' (Isalan et al. (2001) Biochemistry 40, 830-836). Here, we report that Gq1 is able to arrest the action of a DNA polymerase on a template-containing telomeric sequence. Inhibition occurs in a concentration-dependent manner, probably by forming a stabilized G-quadruplex.protein complex. Furthermore, Gq1 inhibits the apparent activity of the enzyme telomerase in vitro, with an IC(50) value of 74.3 +/- 11.1 nM. Possible molecular mechanisms of inhibition are discussed, together with the potential for using engineered zinc fingers to interfere with the cellular processes associated with telomere function.     

Molecular cloning and characterization of a cDNA encoding a novel cuticle protein in the silkworm,Bombyx mori

We have cloned the full length of a novel cDNA named Bombyx mori cuticle protein that contains an AlaAlaProAla/Val-repeat (BMCPA) from a cDNA library of integument in the larval silkworm. Both a typical tandem repeat (A-A-P-A/V) for cuticle protein and a unique tandem repeat with Ser, Ala, Gly, Pro, Val, Tyr and Thr were observed in the predicted amino acid sequence of the cDNA encoding BMCPA. Approximately 80% of the amino acids in BMCPA were composed of Ser, Ala, Gly, Pro, Val and Tyr. Northern-hybridization analysis indicated that BMCPA mRNA is expressed only in the larval epidermis and that the expression pattern of the BMCPA gene in the developmental stage was observed mainly at the larval stage. We propose BMCPA may be a novel component of cuticle, and may play an important role in the integument of the larval silkworm.