We have examined the transcription in a cell-free system of all the members of a highly regulated gene family, the B-like hemoglobin genes of the goat. These five genes, which code for embryonic, fetal, juvenile, and adult 0-globin proteins, are all transcribed to roughly the same extent in the in vitro system. In all cases initiation of transcription is accurate. However, two goat 8-globin pseudogenes, as well as several artificially constructed deletion mutants, are not transcribed in vitro. A common feature of the transcriptionally inactive genes is the lack of anAT-rich consensus sequence just upstream of the presumptive initiation site. INTRODUCTION An understanding of the mechanisms which control transcription will be fundamental in explaining the overall process of gene regulation. One promising approach to this problem involves the reconstitution of biologically meaningful transcription events using purified components. We have recently described a transcription system, consisting of a cultured cell extract (S100) and purified RNA polymerase II, which will initiate transcription on cloned DNA templates at the same sites used for initiation in vivo (1,2). Another in vitro transcription system has subsequently been described, consisting of whole cell extracts independent of exogenous RNA polymerase II (3). Both viral (1,3-6) and chromosomal (2,4,7-9) genes are accurately transcribed by these methods. Given the availability of soluble factors which will mediate accurate transcription initiation in vitro, two kinds of questions may be immediately posed: first, do the transcription factors in the extracts discriminate among promoters in a way that reflects in vivo regulatory events, and second, what are the specific DNA sequences near the site of transcription initiation which are required for promoter recognition? In order to begin to address these questions, we have studied the transcription of the entire family of s-like globin genes of the goat in the S100 system. This gene family has been described in detail previously (10-13); it consists five developmentally regulated sequences: sI and £1I (presumptive embryonic), y C IRL Press Umited, 1 Falconberg Court, London W1V 5FG, U.K. Nucleic Acids Research Volume 9 Number 17 1981