Initiation of simian virus 40 (SV40) DNA replication is facilitated by two auxiliary sequences that flank the minimally required origin (on) core sequence. In monkey cells, the replication rate of each of the four on configurations changed with time after transfection in a characteristic pattern. This pattern was reproduced in an extract from SV40-infected monkey cells by varying the ratio of DNA substrate to cell extract; DNA replication in vitro depended on ori auxiliary sequences to the same extent as they did in vivo. Facilitation by ori auxiliary sequences was lost at high ratios of DNA to cell extract, revealing that the activity of these sequences required either multiple initiation factors or a molar excess of one initiation factor bound to ori. This parameter, together with ionic strength and the method used to measure DNA replication, determined the level of facilitation by on auxiliary sequences in vitro. The activity of ori auxiliary sequences was not diminished in vivo or in vitro by increasing amounts of large tumor antigen. Therefore, ori auxiliary sequences promoted initiation of replication at some step after tumor antigen binding to on. Furthermore, although cellular factors could modulate the activity of on auxiliary sequences in vitro, these factors did not appear to involve nucleosome assembly because no correlation was observed between the number of nucleosomes assembled per DNA molecule and facilitation by on auxiliary sequences. These results demonstrate that SV40 on auxiliary sequences can function in vitro as they do in vivo and begin to elucidate their role in initiating DNA replication.Genetically defined cis-acting sequences that function as origins of DNA replication (ori) in eucaryotic genomes frequently contain two primary components: a core component that is dedicated to DNA replication and an auxiliary component, containing transcription promoter or enhancer elements, that may be involved in both transcription and replication (reviewed in reference 13). ori core is required for replication under all conditions and determines where DNA synthesis begins. However, ori auxiliary sequences are dispensable under some conditions. In the case of simian virus 40 (SV40), ori consists of three components: ori core, aiux-1, and aiux-2 (Fig. 1). When ori interacts with SV40 large tumor antigen (T-Ag) in the presence of permissive cell factors, bidirectional DNA replication originates at the junction of ori core and T-Ag binding region I (12,14,21).auix-J and aux-2 are noninterchangeable sequences that flank ori core and facilitate its activity in vivo (1,10,23,27,29). Deletion of aiux-J reduces replication about sixfold.Deletion of the 72-base-pair (bp) repeats (enhancers) has no effect on DNA replication as long as three or more of the six G3 qCG, motifs that make up part of the promoter are present (aiux-2). Elimination of all G3_CG, motifs reduces

A total of 42,160 individuals were typed for HLA-A and HLA-B by both serology and PCR-based typing. The HLA assignments included all of the known serological equivalents. The majority of the individuals (99.9%) were from U.S. minority population groups. The serologic typing was performed between 1993 and 1997 at the time of recruitment for the National Bone Marrow Program (NMDP) registry. The polymerase chain reaction (PCR)-based typing was carried out in two phases. In phase I, DNA typing was performed by PCR using sequence-specific oligonucleotide probes (PCR-SSOP) or PCR using sequence-specific primers (PCR-SSP) without knowledge of the serologic assignments. Discrepancies were identified between the serologic and DNA assignments in 24% of the volunteers (8% of volunteers differed for only HLA-A assignments, 13% for HLA-B, and 3% for both HLA-A and -B) and a potential explanation was assigned each discrepant serology/DNA pair. In phase II, a random sampling scheme was used to select a statistically significant number of individuals for repeat DNA typing from each of these categories. The categories included antigens missed by serology, nonexpressed (null) alleles, PCR amplification failures, misassignment of antigens and nomenclature issues. Only a single individual was found to carry a null allele. DNA-based testing correctly typed nearly 99% of the donors at HLA-A, more than 98% at HLA-B, and more than 97% at both HLA-A and -B validating this methodology for registry typing.

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DNA-based typing of HLA class I alleles of the HLA-A and HLA-B loci using sequence-specific oligonucleotide primers and/or probes has been used for the large-scale typing of individuals for the National Marrow Donor Program unrelated donor registry. Typing was performed by 16 laboratories at a low level of resolution (e.g. A*01, B*07). The results of blinded quality control analysis for the first 12 months of the project show the typing to be highly accurate, specific and reliable. The total error rate based on 11,545 HLA-A and 11,428 HLA-B assignments was 1.1% for HLA-A and 1.9% for HLA-B. This level of accuracy is particularly remarkable because the quality control samples could not be distinguished from 64,180 donor samples tested at the same time by the laboratories.

SV40 origin auxiliary sequence 1 (aux-1) encompasses T-antigen (T-ag) binding site I and facilitates origin core (ori-core) activity in whole cells or cell extracts. Aux-1 activity depended completely upon its sequence, orientation and spacing relative to ori-core. Aux-1 activity was lost either by inserting 10 base pairs between aux-1 and ori-core or by placing either orientation of aux-1 on the opposite side of ori-core. Reversing the orientation of aux-1 in its normal position actually inhibited replication. Easily unwound DNA sequences that stimulate yeast or E. coli origins of replication could not replace aux-1. Aux-1 did not affect bidirectional replication. Replication remained bidirectional even when aux-1 was inactivated, and deletion of aux-1 did not affect selection of RNA-primed DNA synthesis initiation sites in the origin region: the transition from discontinuous to continuous DNA synthesis that marks the origin of bidirectional replication occurred at the same nucleotide locations in both wild-type and aux-1 deleted origins. These results support a model for initiation of SV40 DNA replication in which T-ag binding to aux-1 (T-ag binding site I) facilitates the efficiency with which T-ag initiates replication at ori-core (T-ag binding site II) without affecting the mechanism by which initiation of DNA replication occurs.