Inactivated Allele Research Articles

Early B-cell Factor 1 Regulates the Expansion of B-cell Progenitors in a Dose-dependent Manner

Transcription factor doses are of importance for normal and malignant B-lymphocyte development; however, the understanding of underlying mechanisms and functional consequences of reduced transcription factor levels is limited. We have analyzed progenitor and B-lineage compartments in mice carrying heterozygote mutations in the E2a, Ebf1, or Pax5 gene. Although lymphoid progenitors from Ebf1 or Pax5 heterozygote mice were specified and lineage-restricted in a manner comparable with Wt progenitors, this process was severely impaired in E2a heterozygote mutant mice. This defect was not significantly enhanced upon combined deletion of E2a with Ebf1 or Pax5. Analysis of the pre-B-cell compartment in Ebf1 heterozygote mice revealed a reduction in cell numbers. These cells expressed Pax5 and other B-lineage-associated genes, and global gene expression analysis suggested that the reduction of the pre-B-cell compartment was a result of impaired pre-B-cell expansion. This idea was supported by a reduction in IL2Rα-expressing late pre-B-cells as well as by cell cycle analysis and by the finding that the complexity of the VDJ rearrangement patterns was comparable in Wt and Ebf1(+/-) pre-B-cells, although the number of progenitors was reduced. Heterozygote deletion of Ebf1 resulted in impaired response to IL7 in vitro and reduced expression levels of pre-BCR on the cell surface, providing possible explanations for the observed stage-specific reduction in cellular expansion. Thus, transcription factor doses are critical for specification as well as expansion of B-lymphoid progenitors, providing increased insight into the molecular regulation of B-cell development.

Normal Dendritic Cell Mobilization to Lymph Nodes under Conditions of Severe Lymphatic Hypoplasia

To address the requirement for lymphatic capillaries in dendritic cell (DC) mobilization from skin to lymph nodes (LNs), we used mice bearing one inactivated allele of vascular endothelial growth factor receptor 3 (VEGFR3) where skin lymphatic capillaries are reported absent. Unexpectedly, DC mobilization from the back skin to draining LNs was similar in magnitude, and kinetics to control mice and humoral immunity appeared intact. By contrast, DC migration from body extremities, including ear and forepaws, was ablated. An evaluation in different regions of skin revealed rare patches of lymphatic capillaries only in body trunk areas where migration was intact. That is, whereas the ear skin was totally devoid of lymphatic capillaries, residual capillaries in the back skin were present though retained only at ∼10% normal density. This reduction in density markedly reduced the clearance of soluble tracers, indicating that normal cell migration was spared under conditions when lymphatic transport function was poor. Residual lymphatic capillaries expressed slightly higher levels of CCL21 and migration of skin DCs to LNs remained dependent on CCR7 in Chy mice. DC migration from the ear could be rescued by the introduction of a limited number of lymphatic capillaries through skin transplantation. Thus, the development of lymphatic capillaries in the skin of body extremities was more severely impacted by a mutant copy of VEGFR3 than trunk skin, but lymphatic transport function was markedly reduced throughout the skin, demonstrating that even under conditions when a marked loss in lymphatic capillary density reduces lymph transport, DC migration from skin to LNs remains normal.

Independent clonal origin of multiple uterine leiomyomas that was determined by X chromosome inactivation and microsatellite analysis

In an attempt to clarify the clonality and genetic relationships that are involved in the tumorigenesis of uterine leiomyomas, we used a total of 43 multiple leiomyomas from 14 patients and analyzed the allelic status with 15 microsatellite markers and X chromosome inactivation analysis. We have used a set of 15 microsatellite polymorphism markers mapped on 3q, 7p, 11, and 15q by automated analysis. The X chromosome inactivation was evaluated by the methylation status of the X-linked androgen receptor gene. Loss of heterozygosity analysis showed a different pattern in 7 of the 8 cases with allelic loss for at least 1 of 15 microsatellite markers that were analyzed. A similar loss of heterozygosity findings at 7p22-15 was detected in 3 samples from the same patient. X chromosome inactivation analysis demonstrated the same inactivated allele in all tumors of the 9 of 12 informative patients; different inactivation patterns were observed in 3 cases. Our data support the concept that uterine leiomyomas are derived from a single cell but are generated independently in the uterus. Loss of heterozygosity findings at 7p22-15 are consistent with previous data that suggested the relevance of chromosomal aberrations at 7p that were involved in individual uterine leiomyomas.

Hepatocellular carcinoma associated with focal nodular hyperplasia. Report of a case with clonal analysis.

We describe a hepatocellular carcinoma partially surrounded by focal nodular hyperplasia in a 65-year-old female patient. In order to clarify the relationship of the hepatocellular carcinoma and the adjacent focal nodular hyperplasia, clonal analysis was conducted. The clonal analysis was based on the methylation pattern of the polymorphic X-chromosome-linked androgen receptor gene (HUMARA). The allelic bands from the amplification of the focal nodular hyperplasia and of the hepatocellular carcinoma showed a significant reduction in the intensity of one of the two alleles as compared with two alleles of equal intensity in the buff coat after HhaI digestion, which indicated that these two parts were monoclonal. However, the inactivated allele in the focal nodular hyperplasia and that in the hepatocellular carcinoma were not identical. Therefore, the focal nodular hyperplasia and hepatocellular carcinoma probably derived from the clonal expansion of two different clones.

Clonal analysis of superficial depressed-type gastric carcinoma in humans.

An important unanswered question concerning the histogenesis of superficial-type gastric carcinoma is whether it is monoclonal or multiclonal in origin. Therefore, the authors analyzed multiple areas of each cancer with a clonality assay based on trinucleotide repeat length polymorphism of the human androgen receptor gene (HUMARA) that was subject to random inactivation of X chromosomes. The HUMARA assay was applied to 15 gastric carcinomas, early and advanced stage, manifested in superficial, depressed lesions of various sizes and at least some signet ring cells. DNA was extracted from fresh frozen and formalin fixed tumor tissues that were microdissected from the mucosal lesions, and the HUMARA locus was amplified by polymerase chain reaction with and without prior digestion of nonmethylated DNA with Hpa II. The amplified DNA samples were loaded on polyacrylamide gels, electrophoresed, and visualized by a silver-staining method. In the 15 cases examined, 9 cancers were informative (had features of the types sought in this study), and in these 9 cancers a total of 57 areas were analyzed. In 7 of the 9 cancers, the inactivated allele was common to all the informative areas of each tumor, irrespective of the macroscopic shape of the tumor or the degree of histologic heterogeneity within it. In one of the two remaining cancers, the inactivated allele of one of the areas examined was different from those in the other areas. Most of the superficial depressed-type gastric carcinomas in this study were demonstrated to be of monoclonal origin. This finding supports a notion expressed previously in the literature that superficial-type carcinoma has a long natural history, and it indicates that efforts to detect gastric carcinomas in early stages to improve patients' survival should be encouraged.

Double replacement gene targeting for the production of a series of mouse strains with different prion protein gene alterations.

We have developed a double replacement gene targeting strategy which enables the production of a series of mouse strains bearing different subtle alterations to endogenous genes. This is a two-step process in which a region of the gene of interest is first replaced with a selectable marker to produce an inactivated allele, which is then re-targeted with a second vector to reconstruct the inactivated allele, concomitantly introducing an engineered mutation. Five independent embryonic stem cell lines have been produced bearing different targeted alterations to the prion protein gene, including one which raises the level of expression. We have constructed mice bearing the codon 101 proline to leucine substitution linked to the human familial prion disease, Gerstmann-Straussler-Scheinker syndrome. We anticipate that this procedure will have applications to the study of human inherited diseases and the development of therapies.

Sequence and inactivation of the pss gene of Escherichia coli. Phosphatidylethanolamine may not be essential for cell viability

Phosphatidylethanolamine is the only zwitterionic phospholipid in Escherichia coli and accounts for 70-80% of the total glycerophospholipids of this organism. To investigate the function of phosphatidylethanolamine in E. coli, we constructed an inactivated allele (pss93::kan) of the gene encoding the phosphatidylserine synthase which catalyzes the committed step to the synthesis of phosphatidylethanolamine. Growth of this mutant was dependent on a plasmid-borne copy of the wild type gene. After curing the mutant of the wild type gene, growth stopped when the content of phosphatidylethanolamine reached 30% of the total phospholipid. Divalent metal ions at millimolar concentrations suppressed the growth phenotype of the mutant in the following order of efficiency: Ca2+ greater than Mg2+ greater than Sr2+. Although phosphatidylserine synthase activity was not detectable, phosphatidylethanolamine was still present at 0.007% of the total phospholipid after growth for many generations in rich medium containing 20 mM Mg2+. The remainder of the phospholipid was primarily phosphatidylglycerol and cardiolipin with no other unique phosphate-containing chloroform-soluble material present. The phospholipid to protein ratio and the fatty acid composition were very similar to the parental strain. The broad divalent metal ion auxotrophy brought about by the lack of phosphatidylethanolamine suggests a primarily structural role for this phospholipid in E. coli.

Sequence and inactivation of the pss gene of Escherichia coli. Phosphatidylethanolamine may not be essential for cell viability.

Phosphatidylethanolamine is the only zwitterionic phospholipid in Escherichia coli and accounts for 70-80% of the total glycerophospholipids of this organism. To investigate the function of phosphatidylethanolamine in E. coli, we constructed an inactivated allele (pss93::kan) of the gene encoding the phosphatidylserine synthase which catalyzes the committed step to the synthesis of phosphatidylethanolamine. Growth of this mutant was dependent on a plasmid-borne copy of the wild type gene. After curing the mutant of the wild type gene, growth stopped when the content of phosphatidylethanolamine reached 30% of the total phospholipid. Divalent metal ions at millimolar concentrations suppressed the growth phenotype of the mutant in the following order of efficiency: Ca2+ greater than Mg2+ greater than Sr2+. Although phosphatidylserine synthase activity was not detectable, phosphatidylethanolamine was still present at 0.007% of the total phospholipid after growth for many generations in rich medium containing 20 mM Mg2+. The remainder of the phospholipid was primarily phosphatidylglycerol and cardiolipin with no other unique phosphate-containing chloroform-soluble material present. The phospholipid to protein ratio and the fatty acid composition were very similar to the parental strain. The broad divalent metal ion auxotrophy brought about by the lack of phosphatidylethanolamine suggests a primarily structural role for this phospholipid in E. coli.