(A) Schematic representation of murine 4-Ptase I cDNA clones. (Upper) The assembled 4-Ptase I α cDNA is depicted by the full open box. (Lower) The location of the initial clone obtained from subtraction analysis; the phage clone that contained a poly(A) tail and lastly a composite cDNA of clones obtained by RT-PCR (shaded box corresponds to the coding region). The locations of the oligonucleotide primers (A–E, H, and I) used to clone the cDNA and oligonucleotide primers F and G used in the RT-PCR to document expression of 4-Ptase I in normal and GATA-1^- megakaryocytes are shown. (B, Upper) mRNA expression of 4-Ptase I in normal (+/) and GATA-1⁻ (−/) megakaryocytes. cDNA from these two megakaryocyte populations was used in a PCR. Oligonucleotide primers F and G (see A for positions) in the 3′ end of the coding region of mouse 4-Ptase I were used. The amount of input cDNA was normalized with respect to HPRT expression as shown. Numbers of PCR cycles performed to obtain the cDNA product are shown below each lane. (Lower) Western blot analysis of 4-Ptase I protein expression from normal (+/) and GATA-1⁻ (−/) primary fetal liver cells containing ≈ 70% megakaryocytes. Above, 4-Ptase I protein was detected, as a doublet, by antibody to the C terminal of 4-Ptase I. This antibody reacts with the C termini of both 4-Ptase I and II. Below, the same blot was stripped and reprobed with an antibody to c-src to document protein loading in all of the lanes.

(A) Growth curves of primary megakaryocytes from GATA-1⁻ progenitors. Progenitors infected with one of two different retroviruses, one containing human 4-Ptase I and GFP and the other just GFP on its own. Selected progenitors then were expanded in liquid culture with 1% recombinant thrombopoietin-conditioned tissue culture medium. The number of cells with acetylcholinesterase activity in 20-μl aliquots of the culture is shown as a function of the duration of culture. The values shown are an average ± 1 SD from three experiments. (B) Human 4-Ptase I expression was assayed by RT-PCR in aliquots of the two retrovirally infected GATA-1⁻ cultures in A. (Upper) Human 4-Ptase I expression was assayed, and the input amount of cDNA was normalized with respect to HPRT expression (Lower). The number of PCR cycles is shown at the bottom. (C) Growth curves of primary megakaryocytes from retrovirally infected progenitors of normal littermate embryos of GATA-1⁻ embryos. The growth curves were performed in the same manner as A. Note the different ordinates in A versus C. (D) Human 4-Ptase I expression was assayed by RT-PCR in aliquots of the two retrovirally infected cultures of normal progenitors in C. The RT-PCR analysis is illustrated as in B.

Equal numbers of GATA-1⁻ hematopoietic progenitors were infected with retroviruses containing either empty retrovirus (vector alone), the puromycin selection marker [−/(puro)], or human 4-Ptase I plus the puromycin-resistance gene [−/(4-Ptase + Puro)]. In each experiment equal numbers of lineage-depleted GATA-1⁻ fetal liver cells (1–5 × 10⁵) were infected for 48 h with the retroviral supernatants indicated. The cells then were plated in methylcellulose containing 1% recombinant thrombopoietin conditioned tissue culture medium with 1 μg/ml puromycin. The number and size of the colonies was assessed 5–6 days after plating. The numbers of abnormal and normal size colony from three independent experiments are shown. (Lower) Plates of GATA-1⁻ mutant progenitors infected with [−/(4-Ptase + Puro)] or without [−/(puro)] 4-Ptase I are shown. At this magnification (×2), only the abnormal-size megakaryocyte colonies can be seen and are present only in the −/(puro) plate (indicated by the arrows). Infection with 4-Ptase I abolishes growth of these colonies in the −/(4-Ptase + Puro) plate.

(A) Effect of 4-Ptase I overexpression on the proliferation of NIH 3T3 cells. Time course for the growth of NIH 3T3 cells infected with control retrovirus (●) or retrovirus overexpressing 4-Ptase I (■). Points represent the average of three values. Error bars indicate the range of values measured for each time point. 4-Ptase I overexpression does not enhance apoptosis of NIH 3T3 cells. (B) FACS analysis of the DNA content of NIH 3T3 cells infected with control retrovirus 24 h after apoptosis induction by trypsin treatment. The arrow indicates the population of apoptotic cells with DNA content less than G₁ cells. (C) FACS analysis of the DNA content of NIH 3T3 cells infected with control retrovirus (solid line) or retrovirus overexpressing 4-Ptase I (dashed line).