Elsevier

Methods in Enzymology

Volume 200, 1991, Pages 533-546
Methods in Enzymology

[45] Cloning members of protein-tyrosine kinase family using polymerase chain reaction

https://doi.org/10.1016/0076-6879(91)00169-WGet rights and content

Publisher Summary

This chapter presents the process of cloning members of protein-tryosine kinase family by using polymerase chain reaction (PCR). The PCR-based approach requires both the presence of annealing sites for the oligonucleotide primers, and the further constraint of their proximity and correct orientation with respect to each other. This makes it possible to trade the gains in specificity derived from these additional constraints for considerable leeway in the degeneracy of the PCR primers employed. This PCR-based cloning protocol has already proved highly effective in revealing previously unknown members of the protein-tyrosine kinase family. The first step in the selection of primer sequences is the generation of an amino acid sequence alignment of the feature conserved in the catalytic domain of the protein-tyrosine kinase family. The best amino acid motifs for generation of PCR primers are those most highly conserved among the known protein family members. When the amino acid sequences of the catalytic domains of the protein tyrosine kinases are aligned, these conserved sequence motifs become easily apparent. As with the selection of any degenerate oligonucleotide sequence for library screening, sequences with minimal codon degeneracy should be selected, although it is possible (indeed usual) that the sequence motifs available for primer generation do not afford this luxury.

References (44)

  • E. Shtivelman et al.

    Cell (Cambridge, Mass.)

    (1986)
  • J.D. Marth et al.

    Cell (Cambridge, Mass.)

    (1985)
  • Y. Ebina et al.

    Cell (Cambridge, Mass.)

    (1985)
  • A.F. Wilks et al.

    Gene

    (1989)
  • S.K. Hanks et al.

    Science

    (1988)
  • K. Mullis et al.
  • R.K. Saiki et al.

    Science

    (1985)
  • R.K. Saiki et al.

    Science

    (1988)
  • A.F. Wilks
  • Y. Ben-Neriah et al.

    Nature (London)

    (1988)
  • Q-L. Hao et al.

    Mol. Cell. Biol.

    (1989)
  • A.F. Wilks et al.

    Oncogene

    (1988)
  • G.D. Kruh et al.

    Science

    (1986)
  • J. Sukegawa et al.

    Mol. Cell. Biol.

    (1987)
  • S. Katamine et al.

    Mol. Cell. Biol.

    (1988)
  • Y. Yamanishi et al.

    Mol. Cell. Biol.

    (1987)
  • D. Holtzman et al.
  • K. Strebhart et al.
  • R.J. Gregory et al.

    Mol. Cell. Biol.

    (1987)
  • A. Ullrich et al.

    EMBO J.

    (1986)
  • H. Matsushime et al.

    Mol. Cell. Biol.

    (1986)
  • D.D.L. Bowtell et al.

    Genes Dev.

    (1988)
  • Cited by (62)

    • The JAK–STAT–SOCS Signaling Cascade

      2022, Encyclopedia of Cell Biology: Volume 1-6, Second Edition
    • The JAK-STAT-SOCS Signaling Cascade

      2016, Encyclopedia of Cell Biology
    • Cloning, expression and characterization of a gene encoding mitogen activated protein kinase 2 (MPK2) from tetrahymena thermophila

      2014, Gene
      Citation Excerpt :

      GenBank accession numbers of MAPK sequences and their initials used in constructing the tree are as follows: ATMPK7_Ath (BAA04870), BMK1A_Hsa (AAA82931), ERKA_Ddi (AAA59387), ERK1/2_Clu (EEQ37361), ERK1/2_Cme (BAM82886), ERK1/2_Spu (NP_999813), ERK1_Ddi (AAA59387), ERK1_Hsa (P27361), ERK2_Hsa (P28482), ERK5_Hsa (AAA81381), ERK7_Dme (NP_727335), ERK7_Dre (ABC94475), ERK7_Rno (NP_775453), ERK8_Hsa (NP_620590), FUS3_Sce (AAA34613), Fus3_Act (EAW06909), Fus3_Afv (EED56145), HOG1_Clu (EEQ37760), HOG1_Ctp (EER34321), HOG1_Lel (EDK43139), HOG1_Sce (AAA34680), HOG1p_Sce (AAB67558), JNK1A2_Hsa (AAC50607), JNK2A1_Hsa (AAC50606), JNK_Ame (XP_392806), JNK_Dme (AAC47325), KSS1_Sce (P14681), MAPK15_Hsa (NP_620590), MAPK6_Osa (ACD76439), MAPK15_Hsa (NP_620590), NTF3_Nta (CAA49592), osMAPK_Ppa (XP_002489575), osMAPK_Bfu (A1IVT7), p38G_Hsa (AAB40118), p38B1_Hsa (AAB05036), p38_Cgr (Q6FIU2), p38_Kaf (CCF56594), p38_Kla (Q6CJA8), p38_Yli (Q6C4M9), p43_Nta (CAA58760), p45_Nta (CAA58761), p97mapk_Hsa (CAA56709), SAPK1A_Hsa (P45984), SAPK3_Hsa (P53778.3), SIMAPK_Zma (NP_001105239), SIPK_Natt (ABJ89812), SLT2_Sce_(CAA41954), SPK1_Spo (CAA40610), STY1_Ssl (EDN91730), STY1_Ncr (Q96TL5), STY1_Spo (CAA61537), SUR1_Ce(lAAA18956), TtMPK1 (XP_001025119), TtMPK2 (XP_001031635), TtMPK3 (XP_001020076), WIPK_Nta (BAA09600). Using RT-PCR with degenerate primers coupled to RACE cloning strategy (Hanks and Linberg, 1991; Wilks, 1991), the forward poly dC18 primer and a reverse degenerate 3′ERK primer set yielded PCR product of about 740 bp. The 740 bp fragments were cloned in pGEM-T Easy vector, and manually sequenced to screen the individual clones.

    • Investigation of JAK1 and STAT3 polymorphisms and their gene-gene interactions in nonspecific digestive disorder of rabbits

      2014, Gene
      Citation Excerpt :

      Consequently, it is time to identify the genetic markers associated with NSDD in rabbits. Janus kinases (JAKs) are nonreceptor tyrosine kinases, discovered in the search for novel protein tyrosine kinases using PCR-based strategies or low-stringency hybridization (Fwilks, 1991; Kawamura et al., 1994; Krolewski et al., 1990; Wilks, 1989). In mammals, there are four members of the family (JAK1, JAK2, JAK3, and TYK2).

    • The structural basis of Janus kinase 2 inhibition by a potent and specific pan-Janus kinase inhibitor

      2006, Blood
      Citation Excerpt :

      The Janus kinases (JAKs) are an important family of intracellular protein tyrosine kinases (PTKs), with 4 mammalian members, JAK1, JAK2, JAK3, and TYK2,1-5 as well as homologs in chicken,6 fish,7 and Drosophila.8

    View all citing articles on Scopus
    View full text