We used a sensitive in vitro culture system to follow, in embryonic blood, the number and state of commitment of B cell precursors along ontogeny. We describe a wave of circulating multipotent progenitors, first detectable at day 10 of gestation, and reaching a maximum in absolute numbers at day 12. They are undetectable by day 14 of gestation, when committed B cell precursors can be detected in fetal liver. Embryonic marrow contains B cell progenitors by day 15. We propose that fetal liver, thymus, and bone marrow are colonized by the same wave of multipotent hematopoietic cells and define embryonic blood at day 11 of gestation as an important source of multipotent hematopoietic cells, virtually deprived of committed and mature contaminants.

In vitro enzymatic amplification of nucleic acids by PCR or other techniques is a very sensitive method to detect rare DNA segments. We present here a protocol that allows the rapid, sensitive and precise quantification of DNA molecules using PCR amplification run to saturation. The DNA (or cDNA) to be assayed is co-amplified with known amounts of an internal standard DNA. We show that the latter must be almost identical to the assayed DNA, otherwise quantification at the plateau is unreliable. The read-out of the amplification involves one or two additional oligonucleotides. Using fluorescent oligonucleotides as primers in run-off reactions together with an automated DNA sequencer, we could measure the level of expression of several genes, like the murine MHC class I H-2Kd or a specific T cell receptor beta chain transcript in the course of an immunization. mRNA levels were normalized by measuring in a similar manner the number of transcripts encoding the housekeeping gene HPRT. Finally, our procedure might allow the rapid analysis of a large number of samples at the same time, as illustrated by the simultaneous analysis of the mRNAs encoding the CD4 and CD8 murine T cell markers.

Two of the first human immunodeficiency virus type-1 (HIV-1) strains isolated were authenticated by reanalyzing original cultured samples stored at the Collection Nationale de Culture des Microorganismes as well as uncultured primary material. Cloned polymerase chain reaction products were used to analyze coding sequences of the V3 loop in the gp120 glycoprotein. The original isolate HIV-1 Bru, formerly called LAV, was derived from patient BRU. HIV-1 Lai was derived from patient LAI and contaminated a HIV-1 Bru culture between 20 July and 3 August 1983. The culture became, in effect, HIV-1 Lai, identifiable by a unique motif in the V3 loop. Because of this contamination two, rather than one, HIV-1 isolates were sent to the Laboratory of Tumor Cell Biology at the National Cancer Institute on 23 September 1983. Original HIV-1 Bru was indeed present in the sample marked JBB/LAV. However the M2T-/B sample harbored HIV-1 Lai, a strain capable of growing on established cell lines. The striking similarity between HIV-1 Lai (formerly LAV-Bru) and HTLV-3B sequences remains.

The evolution of an 851-bp segment of the human immunodeficiency virus type 1 (HIV-1) genome encoding the nef open reading frame and U3/R elements of the long terminal repeat has been followed over a 4-year period in vivo and in vitro. The population of viral sequences at any given time was established by sequencing cloned polymerase chain reaction products. The samples studied were derived from the same man for whom a detailed analysis of the tat gene was previously described (A