initial cdna  (OriGene)

Journal: The Journal of biological chemistry

Article Title: Cloning and expression of sialidase L, a NeuAcalpha2-->3Gal-specific sialidase from the leech, Macrobdella decora.

doi: 10.1074/jbc.271.32.19219

Figure Lengend Snippet: FIG. 1. Schematic representation of sialidase L cDNA and the DNA fragments produced by PCR from leech total RNA. A, re- striction map of sialidase L cDNA. Open box, the open reading frame; hatched boxes, the conserved consensus repeats (Asp boxes). B, PCR fragments obtained by RT-PCR (fragment A), 59-RACE (fragments B and C), and RLM-RACE (fragment D) from the leech total RNA as described under “Experimental Procedures.” The molecular size and the nucleotide sequence of each fragment are scaled according to the siali- dase L cDNA map in A. The primers used for amplifying the PCR fragments are indicated on both ends of each fragment.

Article Snippet: RESULTS AND DISCUSSION Isolation of a cDNA Encoding Sialidase L—Initially, the M. decora leech cDNA library constructed in lZAP II system (Stratagene) was screened with the DNA probe derived from the RT-PCR product (fragment A) of sialidase L. By screening approximately 2 3 106 phages from the leech cDNA library, no positive clone was detected.

Techniques: Produced, Reverse Transcription Polymerase Chain Reaction, Sequencing

Journal: The Journal of biological chemistry

Article Title: Cloning and expression of sialidase L, a NeuAcalpha2-->3Gal-specific sialidase from the leech, Macrobdella decora.

doi: 10.1074/jbc.271.32.19219

Figure Lengend Snippet: FIG. 2. cDNA and deduced amino acid sequences of sialidase L. The de- duced amino acid sequence in the single- letter code is shown under the nucleotide sequence. The peptide sequences obtained from the native protein by CNBr cleavage or tryptic digestion are underlined and named according to the previous report (5). The Asp boxes are shaded and the FRIP region is boxed. In-frame stop codons are marked with asterisks.

Article Snippet: RESULTS AND DISCUSSION Isolation of a cDNA Encoding Sialidase L—Initially, the M. decora leech cDNA library constructed in lZAP II system (Stratagene) was screened with the DNA probe derived from the RT-PCR product (fragment A) of sialidase L. By screening approximately 2 3 106 phages from the leech cDNA library, no positive clone was detected.

Techniques: Sequencing

Journal: The Journal of biological chemistry

Article Title: Cloning and expression of sialidase L, a NeuAcalpha2-->3Gal-specific sialidase from the leech, Macrobdella decora.

doi: 10.1074/jbc.271.32.19219

Figure Lengend Snippet: FIG. 3. Hydropathy profile of the deduced amino acid se- quence of sialidase L. Kyte-Doolittle hydrophobicity profile (26) of sialidase L is plotted with an 11-residue window. The putative signal peptide is indicated by a solid bar.

Article Snippet: RESULTS AND DISCUSSION Isolation of a cDNA Encoding Sialidase L—Initially, the M. decora leech cDNA library constructed in lZAP II system (Stratagene) was screened with the DNA probe derived from the RT-PCR product (fragment A) of sialidase L. By screening approximately 2 3 106 phages from the leech cDNA library, no positive clone was detected.

Techniques: Residue

Journal: The Journal of biological chemistry

Article Title: Cloning and expression of sialidase L, a NeuAcalpha2-->3Gal-specific sialidase from the leech, Macrobdella decora.

doi: 10.1074/jbc.271.32.19219

Figure Lengend Snippet: FIG. 4. SDS-PAGE of the recombinant sialidase L. The samples were run on a 10% SDS-PAGE under reducing conditions and the gel was stained with Coomassie Brilliant Blue. Lane 1, the molecular mass standards (from top to bottom, phosphorylase B, bovine serum albumin, ovalbumin, carbonic anhydrase, and soybean trypsin inhibitor); lane 2, the uninduced cell lysate; lane 3, the IPTG-induced cell lysate; lane 4, the purified recombinant sialidase L after octyl-Sepharose chromatog- raphy. The detailed assay conditions are described under “Experimen- tal Procedures.”

Article Snippet: RESULTS AND DISCUSSION Isolation of a cDNA Encoding Sialidase L—Initially, the M. decora leech cDNA library constructed in lZAP II system (Stratagene) was screened with the DNA probe derived from the RT-PCR product (fragment A) of sialidase L. By screening approximately 2 3 106 phages from the leech cDNA library, no positive clone was detected.

Techniques: SDS Page, Recombinant, Staining, Purification

Journal: The Journal of biological chemistry

Article Title: Cloning and expression of sialidase L, a NeuAcalpha2-->3Gal-specific sialidase from the leech, Macrobdella decora.

doi: 10.1074/jbc.271.32.19219

Figure Lengend Snippet: FIG. 5. TLC showing the hydrolysis of 3*-sialyllactose, 6*-sia- lyllactose, and GD3 by the recombinant sialidase L (rSL) and Clostridial sialidase (CS). Each substrate (8 nmol) was incubated with 10 units of rSL or CS at 37 °C for 6 h. 1, standard 2,7-anhydro- NeuAc (top) and NeuAc (bottom); 2, 39-sialyllactose; 3, 39-sialyllactose 1 CS; 4, 39-sialyllactose 1 rSL; 5, 69-sialyllactose; 6, 69-sialyllactose 1 CS; 7, 69-sialyllactose 1 rSL; 8, GD3; 9, GD3 1 CS; 10, GD3 1 rSL; 11, standard GM3 (NeuAca233Galb134Glcb1–19Cer, top) and lactose (bot- tom). LacCer, lactosylceramide as indicated by an arrow. The detailed assay conditions are described under “Experimental Procedures.”

Article Snippet: RESULTS AND DISCUSSION Isolation of a cDNA Encoding Sialidase L—Initially, the M. decora leech cDNA library constructed in lZAP II system (Stratagene) was screened with the DNA probe derived from the RT-PCR product (fragment A) of sialidase L. By screening approximately 2 3 106 phages from the leech cDNA library, no positive clone was detected.

Techniques: Recombinant, Incubation

Journal: The Journal of biological chemistry

Article Title: Cloning and expression of sialidase L, a NeuAcalpha2-->3Gal-specific sialidase from the leech, Macrobdella decora.

doi: 10.1074/jbc.271.32.19219

Figure Lengend Snippet: FIG. 6. Alignment of the amino acid sequence and the predicted b-strands of sialidase L (SL) with C. septicum sialidase (CS) and S. typhi- murium sialidase (ST). Shaded areas show sequence identities. Gaps are intro- duced to produce optimal alignment (in- dicated by dashed lines). The Asp boxes and the FRIP region are boxed. The con- served single amino acid residues present in the bacterial and protozoan sialidases as reported previously (24) are shown in boldface type. The amino acid residues in- volved in the active site of ST (23) are marked by asterisks. The positions of b-strands as predicted for SL are shown by the hatched bars, and as observed in the crystal structure for the ST are shown by the open bars. Prediction of b-strands was based on the analysis of the sequence by the method of Garnier et al. (30).

Article Snippet: RESULTS AND DISCUSSION Isolation of a cDNA Encoding Sialidase L—Initially, the M. decora leech cDNA library constructed in lZAP II system (Stratagene) was screened with the DNA probe derived from the RT-PCR product (fragment A) of sialidase L. By screening approximately 2 3 106 phages from the leech cDNA library, no positive clone was detected.

Techniques: Sequencing

Journal: eLife

Article Title: MCPH1 inhibits Condensin II during interphase by regulating its SMC2-Kleisin interface

doi: 10.7554/eLife.73348

Figure Lengend Snippet:

Article Snippet: recombinant DNA reagent , Mouse Ncaph2 cDNA , Origene , MC200537 , Initial cDNA used for cloning in pRNA.

Techniques: Plasmid Preparation, Isolation, Transfection, Construct, Produced, Recombinant, Clone Assay, In Vitro, Injection, CRISPR, Sequencing, Imaging, Labeling, Protein Purification, Silver Staining, Staining, Protease Inhibitor, In Vivo, Fluorescence, Amplification, Software