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Little is known about the general biology of minisatellites. The purpose of this study is to examine repeat mutations from the D1S80 minisatellite locus by sequence analysis to elucidate the mutational process at this locus. This is a highly polymorphic minisatellite locus, located in the subtelomeric region of chromosome 1. We have analyzed 90,000 human germline transmission events and found seven (7) mutations at this locus. The D1S80 alleles of the parentage trio, the child, mother, and the alleged father were sequenced and the origin of the mutation was determined. Using American Association of Blood Banks (AABB) guidelines, we found a male mutation rate of 1.04 × 10(-4) and a female mutation rate of 5.18 × 10(-5) with an overall mutation rate of approximately 7.77 × 10(-5). Also, in this study, we found that the identified mutations are in close proximity to the center of the repeat array rather than at the ends of the repeat array. Several studies have examined the mutational mechanisms of the minisatellites according to infinite allele model (IAM) and the one-step stepwise mutation model (SMM). In this study, we found that this locus fits into the one-step mutation model (SMM) mechanism in six out of seven instances similar to STR loci.

HLA-B*5701 testing to provide risk stratification for abacavir hypersensitivity has the potential to reduce incidence of hypersensitivity reactions in susceptible individuals. Early experience with clinical HLA-B*5701 testing of the first 100 specimens, from a large clinical reference laboratory in the United States, is presented. Patient samples were tested using a two-step approach. The first step allowed rapid identification of most HLA-B*5701-negative samples in a high throughput mode. The second step involved resolution of putative positives by DNA sequencing to identify B*5701 specifically as well as other B57 subtypes. Test reporting included a phone call from a genetic counselor to obtain the ethnic background and indication for testing and to provide a patient-specific interpretation. The patients population was comprised of Caucasians, 84%; Hispanics, 13%; and African Americans, 3%. Among the 100 samples tested, 92% were HLA-B*5701-negative and 8% were positive for the HLA-B*5701 allele. All HLA-B*5701 allele positives were identified in Caucasian patients. Where the indication for testing was obtainable (57 patients), pre-abacavir therapy screening was the indication 67% of the time. Clarification of previous suspected history of hypersensitivity was the indication 33% of the time. Among samples tested to help clarify a previous history of hypersensitivity, 16/19 or 84% did not carry the HLA-B*5701 allele whereas 3/19 (16%) were carriers of the HLA-B*5701 allele. Early utilization of HLA-B*5701 testing in community practice was not always consistent with the clinical indications for testing. Post-test communication assisted in providing physician education and interpretation of patient-specific results.

Annually the Medizinische Dienst der Krankenversicherung Westfalen-Lippe (MDK-WL) performs approximately 120,000 nursing care assessments according to the 11th social statute book (SGB XI). A prospective reflection on the amount of expert assessments and the spectrum of services for the Nursing Care Insurance until 2020, was performed in order to establish a long term strategic controlling. The insured party makes the request for nursing care according to their personal estimation when the need for assistance is increasing. To predict the future amount of expert assessments you have to take into consideration the social background of the families in addition to age and gender (there is a clear correlation between age and the need of assistance). The database from nursing care assessments in 2001 was projected on a demographic model for the region of Westfalen-Lippe. The amount of requests correlates as expected with the patients age and increases exponentially. The incidence in the need of care shows relevant gender differences, but taken into consideration the very different age structure, the overall incidence is very similar. Against the background of the current nursing care law, the MDK-WL has to deal with an annual increase in assessments of 2 %. The requests for single persons are extremely often without foundation (55 % not substantially in need of care vs. 35 %). Looking at the requests of couples, it shows that the ones for women are more often unfounded then the ones for men (39 % vs. 32 %). It is necessary to take the development of the amount of single living persons into consideration to achieve more accurate predictions for the amount of assessments.

The frequencies of DRB1*08 alleles within four major United States populations found within a hematopoietic stem cell volunteer donor database were determined by DNA sequencing of over 60 DRB1*08 positive individuals from each group. Seven of 30 known DRB1*08 alleles were identified within this study population (080101, 080201, 080302, 080401, 0806, 0807, and 0811). Each ethnic group was characterized by a different highly prevalent allele: DRB1*080101 in Caucasians; DRB1*080401 in African-Americans; DRB1*080302 in Asians; and DRB1*080201 in Hispanics. The alleles DRB1*080101, DRB1*080201, and DRB1*080401 were present in all four populations. This report also describes five novel DRB1*08 alleles uncovered during routine human leukocyte antigen typing.

One hundred sixty-one DRB1*03 positive individuals from each of five U.S. population groups (Caucasoids, African Americans, Asians/Pacific Islanders, Hispanics, and Native Americans) were randomly selected from a database of 82,979 individuals. DRB1*03 alleles were identified by polymerase chain reaction-sequence-specific oligonucleotide probe typing. A total of six DRB1*03 alleles out of 21 known alleles were detected. DRB1*03011 was the predominant DRB1*03 allele in all populations. Caucasoids were found to be the least diversified; only DRB1*03011 was observed. African Americans carried DRB1*03021 at a high frequency. This allele was observed in three other populations. DRB1*0304 was found in Asians/Pacific Islanders and DRB1*0305, DRB1*0307 and a new allele, DRB1*0316, was found in Hispanics. A subset of individuals was also typed for DRB3 alleles. DRB3*0101, DRB3*0202, and DRB3*0301 were detected and seven DRB1-DRB3 haplotypes were defined. Testing of other individuals not included in the DRB1*03 frequency study identified a variation of a common extended haplotype, A1, B8, DR3, which carries DRB1*0304 and two previously unreported DRB1*03 alleles, DRB1*0311 and *0320, are also described.

The National Marrow Donor Program (NMDP) has instituted an approach to address the impact of new alleles on the DNA-based HLA assignments obtained during volunteer donor typing. This approach was applied to the DRB typing results from 371,187 donors received from 14 laboratories in 1999. Samples were tested with a standardized set of sequence specific oligonucleotide reagents and the positive and negative hybridization results transmitted electronically to the NMDP. A software program interpreted the primary data into HLA assignments and rejected assignments which did not produce a result at the specified level of resolution. Comparison of the HLA assignments derived by the NMDP software to the assignments made by the laboratories using several local software prograins showed 90.5% of the assignments to be identical. Differences in assignments were explained by varying levels of typing resolution, variation in the inclusion of the second expressed DRB loci, disparity arising when alternative assignments were summarized, and failure to submit correct information. When the primary data collected in 1999 were interpreted into HLA assignments using the set of alleles defined in July 2000, 74% of the HLA-DRB assignments were altered by the description of new alleles, justifying the development of this software.

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.

Disruption of intercellular communication (IC) by tumor promoters has been implicated as one of the major events in the promotion process. We studied the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on IC in relation to colony formation (CF) in a coculture system of mouse epidermal JB6 cells, including unpromotable, promotable, and transformed clones. CF was evaluated in cocultures where cells were overlaid onto irradiated mat cells. IC was evaluated by the dye transfer assay in cocultures where overlaid cells were labeled with fluorescent beads. Enhancement of CF by TPA was observed in combinations where promotable clones were used as overlays. However, suppression of IC by TPA was observed in all clones of overlaid cells (day 1) and did not correlate satisfactorily to subsequent CF. Growth-arrested cells retained their capability to communicate with mat cells, while IC between colony-forming cells and mat cells was disrupted during CF (day 5), implying that selective communication is an event secondary to CF. It is suggested that in our experimental model, short-term suppression of IC by TPA may not be sufficient to explain subsequent colony formation and that other factors should be considered.

The possible in vivo role of TGF-beta 1 in regulating various proteins of the extracellular matrix, including fibronectin, collagen I and III, and glycosaminoglycans, was examined by immunohistochemical methods during critical stages of lung morphogenesis in the 11- to 18-day-old mouse embryo. Sections of Bouin-fixed, paraffin-embedded whole embryos were exposed to polyclonal antibodies specific to synthetic peptides present in the precursor part of TGF-beta 1 (pro-TGF-beta 1), in the processed TGF-beta 1 (antibody CC), collagen I and III, fibronectin, followed by the PAP or ABC technique to visualize the location of the antibody. GAG were stained with Alcian Blue 8GX. Our results indicate colocalization of TGF-beta 1 expression and that of matrix proteins in the developing lung when branching morphogenesis (cleft formation) and tissue stabilization occur. The presence of TGF-beta 1 at the epithelial-mesenchymal interfaces of stalks and clefts at a time when matrix proteins can first be visualized in these areas, suggests a direct participation of the growth factor in the development of the basic architecture of the lung.

Transforming growth factors-beta (TGF-beta) are 25 kilodalton (kDa) homodimeric peptides with multifunctional actions controlling the growth, differentiation and function of a broad range of target cells of both epithelial and mesenchymal derivation. They are expressed early in embryogenesis and their tissue-specific and developmentally dependent expression is strongly suggestive of an essential role in particular morphogenetic and histogenetic events. Five distinct TGF-beta s have been characterized so far, with 65-80% homology to each other. By using both molecular biological and immunohistochemical techniques, we are currently attempting to define specific sites of expression of the different TGF-beta s and to determine whether TGF-beta s 1-5 might have unique functions in development and in the mature organism. Comparative study of the promoter regions for the different TGF-beta s and for any particular TGF-beta in different species is also underway. Mechanistically, TGF-beta s act to control gene expression of their target cells, many of their actions converging on a complex, multifaceted scheme of control of matrix proteins and their interactions with cells; these effects on matrix are thought to mediate many of the effects of TGF-beta on development.

We studied gap junctional intercellular communication (IC) in various clones of mouse epidermal JB6 cells and the effect of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) on such communication. JB6 clones used included nonpromotable, promotable, and transformed clones, representing a spectrum in susceptibility to transformation from nontransformed, to initiated (postinitiated), to transformed cells. We used the dye transfer assay and the radioisotope transfer assay, and quantified IC both in homologous pairings, where IC among cells of a single clone was examined, and heterologous pairings, where cells of initiated or transformed clones were paired with cells of a nonpromotable clone. Both pairings showed good IC in the absence of TPA and poor IC in the presence of TPA. However, suppression of IC by TPA was more effective when cells had advanced in promotability. IC was more suppressed by TPA in heterologous pairing than in homologous pairing. These results implied that in advanced stages of promotion, the capability to retain IC with each other (homologous IC) and especially with their nontransformed counterpart (heterologous IC) is progressively lost. Thus we conclude that the interaction of initiated cells and transformed cells with nontransformed cells decreases progressively in this model system for tumor promotion and progression.

We report here that extracellular TGF-beta 1 is associated exclusively with microfibrils of elastin which are present in the extracellular matrix of the inflamed articular joint of the rat. Inflammation was initiated by bacterial cell walls localized in the synovium following intraperitoneal injection of the bacterial components. This synovitis is associated with both destruction of connective tissue components and matrix deposition. The growth factor was localized by using a polyclonal antibody raised to a synthetic peptide corresponding to amino terminal 30 amino acids of TGF-beta 1 in conjunction with a gold-labeled secondary antibody. The results suggest a close association of TGF-beta 1 with proteoglycans which are known to be a major component of the microfibrils in elastin. Proteoglycan-mediated binding and concentration of TGF-beta 1 in specific areas of the extracellular matrix may constitute a mechanism whereby the growth factor could be targeted to specific sites of action.

The relationship of intracytoplasmic type A particles, precursor particles of type B retroviruses, to microtubule organizing centers and to the mitotic apparatus have been studied electronmicroscopically in hamster and murine cell lines in various stages of mitotic arrest by Colcemid and vincristine sulfate. It was shown that the migration of these precursor particles from the cytoplasm to the kinetochore region of chromosomes, occurring in early metaphase, is a function of the inhibition of microtubule formation at the pericentriolar cytocentrum and of the attachment of spindle fibers at one sister kinetochore plate of chromosomes. The association of these type A particles with the mitotic apparatus during Colcemid arrest is reversible by removal of the inhibitor and is inversely proportional to the reattachment of spindle fibers and to the reformation of microtubules. The active participation of microtubules and cytoskeletal proteins in the transport and maturation of type B oncornaviruses is strongly suggested by these findings. Their implications, as to a possible epigenetic mode of transmission of these viruses as well as to the induction of the transformed state are discussed.

A rat liver-derived epithelial cell line transformed with DL-ethionine and the corresponding control cell line were characterized according to morphological and cytochemical criteria to establish their origin from liver epithelium and to identify cellular changes due to transformation by DL-ethionine. The presence of intermediate junctions confirms the epithelial nature; glycogen accumulation and glucose-6-phosphatase activity confirm the hepatic origin of the cells. Persistent alterations resulting from ethionine transformation were variations in cell shape and size, focal multilayered growth, an increase in the nucleolar:nuclear ratio, and a reduction in the number of cells displaying a primary cilium. Hyperplasia of the inner nuclear membrane, elongation and branching of mitochondria, and a reduction in the length and frequency of cell junctions were also characteristic of the transformed cells.