For all experiments, the sense strand was also labeled and used as a control for nonspecific staining
For all experiments, the sense strand was also labeled and used as a control for nonspecific staining. system appears to play a major role in regulating such decisions. 1-4 In humans, studies have shown the Notch pathway to McMMAF be important in hematopoiesis 5-7 and, recently, two Notch pathway elements have been identified as the culprit genes in two McMMAF human congenital syndromes known as Alagille (DELTA and two that are most similar to the protein SERRATE. These genes have been named, respectively, (and (and and clones were originally identified by probing a Stratagene Human Fetal Brain cDNA library with a 32P-dCTP-labeled polymerase chain reaction (PCR) fragment. The PCR probe was made by using degenerate primers (see reference 15 for primer sequences) with high homology to fly and to amplify cDNA from a human placenta library. The fragments were sequenced and determined to contain sequences similar to the 5 ends of the fly ligands. The entire McMMAF cDNA was retrieved as a single insert McMMAF in LambdaZap. Two clones encoding partial sequence of were isolated and rescued from LambdaZap as inserts in pBlueScript sk?. One of these (pBS15) contained 3 kb of sequence and included coding for the start methionine, the other clone was 1.5 kb long and contained sequence internal to pBS15. The 3.5-kb insert from pBS15 was used as a probe to screen a Clontech human fetal brain cDNA library. Two clones of about 3 kb were isolated, one of these (pBS3C2) started at about base 1200 of pBS15 and extended through the polyadenylation signal; the other was internal to it. Together pBS15 and pBS3C2 appeared to contain the McMMAF entire coding sequence of clone was made using nested degenerate PCR primers derived from amino acid sequences in C–1. 16 The primers were ACIATGAA(C/T)AA(C/T)CTIGCIAAC/TTG (sense), AC(A/G)TAIACI GA(C/T)TG(A/G)TA(C/T)TTIGT, and GC(A/G/T)ATIAC(A/G)CA(CT)TC(A/G)TC(C/T)TT(C/T)TC (both antisense) and corresponded to the peptides TMNNLANC, TKYQSVYV, and EKDECVIA, respectively. They were used to amplify DNA from a human genomic clone in a pAC vector. 17 (The pAC clone was identified using low-stringency hybridization with a mouse probe.) The amplified DNA was sequenced for verification, and the larger piece was used to probe a Clontech human fetal brain cDNA library. Three positive clones were identified, and two contained sequence coding for a polypeptide with high homology to mouse and chicken Delta-1. The two clones were slightly overlapping and represented the 3 half of the protein. One of the clones (pBS24) was used to probe a Stratagene human fetal brain cDNA library, and two additional clones were identified. Both contained sequence 5 to pBS24, one of these (pBS18) contained sequence that coded ERK6 for the entire molecule 3 of amino acid 120, based on the mouse and chicken sequences. It included two introns also, the initial intron was located inside the initial EGF do it again and the next was discovered within the 4th EGF do it again. A full-length clone was after that isolated utilizing a probe created from the 5 end of pBS18, from the initial splice site upstream, to rescreen the Stratagene collection. North Blots Two North blots that were bought from Clontech had been probed according with their guidelines. (The same filtration system was employed for analyzing and appearance; the other filtration system was employed for the probe was created from the full-length put of pBS15. For hybridization; the patterns had been identical (data not really shown). Protocols and Probes The process for the hybridizations continues to be described elsewhere. 18 The probe for was transcribed from a 333-bp fragment, coding for the 10th EGF do it again, where there is small homology with was transcribed from a 2-kb cDNA fragment (clone HDL24) encoding the 3 fifty percent of (find above). For any experiments, the feeling strand was also tagged and used being a control for non-specific staining. The control slides were either showed or empty some staining on the edges from the.