A widely recognized difficulty of presently used methods for cDNA cloning is obtaining cDNA segments that contain the entire nucleotide sequence of the corresponding mRNA. The cloning procedure described here mitigates this shortcoming. Of the 105 plasmid-cDNA recombinants obtained per ,ug of rabbit reticulocyte mRNA, about 10%o contained a complete a-or P-globin mRNA sequence, and at least 30 to 50%o, but very likely more, contained the entire globin coding regions. We attribute the high efficiency of cloning full-or nearly fulllength cDNA to (i) the fact that the plasmid DNA vector itself serves as the primer for first-and second-strand cDNA synthesis, (ii) the lack of any nuclease treatment of the products, and (iii) the fact that one of the steps in the procedure results in preferential cloning of recombinants with full-length cDNA's over those with truncated cDNA's.

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SummaryThe T cell receptor. (TCR) junctional regions (N regions) of the common human Vy9 and V62 gene segments were sequenced from the blood and lung of normal individuals (195 transcripts) and a group of individuals with sarcoidosis (220 transcripts), a granulomatous disease in which increased numbers of Vy9+ -y/b+ T cells are often observed. In normal individuals, the vast majority (86%) ofblood Vy9 transcripts used the JyP gene segment. In contrast to this restriction ofJ region usage, there was a large diversity of the junctional region, with <20% of blood Vy9 junctional regions showing identical sequences for any one normal individual. For the blood V62 transcripts in normal individuals, there was restriction of J region usage, with 93% using J61. The junctional regions were even more diverse than for Vy9, with a unique sequence observed in each transcript examined. Compared with blood, sequences from the normal lung showed a small increase in identical junctional regions, particularly in.one individual where 46% of Vy9 transcripts examined were identical, suggesting a response of some y/6 T cells to antigens found in the lung in the normal state . In marked contrast to normals, some individuals with sarcoidosis had large numbers of Vy9 transcripts, as well as VS2 transcripts, sharing identical sequences . For Vy9 blood transcripts, two individuals showed 84 and 56% ofjunctional region sequences to be identical, respectively. Similarly, blood VS2 transcripts showed 43, 33, and 25% identical junctional region sequences in three individuals . In the sarcoid patient with the most striking over-representation of blood Vy9junctional sequences, lung Vy9 transcripts showed increased (67%) use of the same junctional region sequence as in blood. This limited diversity of TCR junctional regions among some individuals with sarcoidosis suggests a response from specific stimuli, possibly antigenic, and that y/6 T cells may play a specific role in granuloma formation in sarcoidosis, as has been suggested in other granulomatous diseases.

To determine whether human airway submucosal glands produce and secrete surfactant proteins, we examined their protein and gene expression in submucosal glands from trachea and bronchi obtained from operated and autopsied lungs within 4 h of death. Using a monoclonal antibody (PE-10) against surfactant protein A (SP-A), a positive immunoperoxidase stain was observed over serous cells of submucosal glands in histologic sections of airway walls. Measurement of SP-A in culture medium samples using single-step enzyme-linked immunosorbent assay showed a significant secretion of SP-A by isolated submucosal glands (1.2 +/- 0.08 ng/ml/h, SEM, n = 40). In gene expression experiments by reverse transciption-polymerase chain reaction, the SP-A complementary DNA (cDNA) segment was amplified from isolated submucosal glands, indicating the presence of SP-A messenger RNA (mRNA) in airway submucosal glands. Bronchial superficial epithelial cells failed to show the presence of SP-A mRNA. No cDNA segment of SP-B, SP-C, or SP-D cDNA was amplified from isolated submucosal glands or superficial epithelial cells, whereas all were amplified from alveolar tissue. Furthermore, in contrast to the control alveolar tissue, which expressed both SP-A1 and SP-A2 genes, SP-A2 gene transcript alone was detected in isolated submucosal glands by Southern analysis that included the digestion of the amplified SP-A cDNA fragment with the restriction enzyme Apa I. These findings indicate that human airway submucosal gland cells can transcribe the SP-A2 gene and produce SP-A protein in a manner different from peripheral airways and alveoli, playing a role in the airway defense mechanism.