Lg Gene Research Articles

Integrating fecal pollution markers and fluorescence analysis for water quality assessment of urban river

Human fecal contamination in urban rivers poses significant health risks, but their potential connections with other substances like dissolved organic matter (DOM) remain underexplored. In this study, five fecal pollution markers related to fecal Bacteroides or human fecal contamination (AllBac, HF183, BacH, Hum2, and Hum163) and DOM along an urban river were analyzed using quantitative polymerase chain reaction (qPCR) and three-dimensional excitation-emission (3D EEM) fluorescence spectrometry. All five markers were detected with average absolute abundance ranging from 2.51 to 6.28 lg gene copies/100 mL, showing a progressive increase along the river (R2 = 0.29–0.92, p < 0.05). Parallel factor analysis identified three dominant DOM components (humic acid-like, fulvic acid-like, and protein-like), with strong positive correlations between protein-like components and all fecal markers (R2 = 0.59–0.66, p < 0.001). Both fecal and DOM distributions consistently showed significant differences between upstream and downstream areas (p < 0.001), suggesting their complementary assessment. While DOM was more sensitive to environmental variables such as rainfall, rubber dam, and tidal dynamic, the combination of fecal pollution markers and 3D EEM analysis allowed a more comprehensive assessment of contamination levels, mitigating potential biases caused by the influence of multiple factors on a single method. Furthermore, due to the strong correlation between protein-like and fecal markers in the DOM, 3D EEM can be used as a pre-detection means for qPCR detection, reducing testing time and costs.

Whole-Genome Sequencing Characterization of Virulence Profiles of Listeria monocytogenes Food and Human Isolates and In Vitro Adhesion/Invasion Assessment

Listeria monocytogenes (Lm) is the causative agent of human listeriosis. Lm strains have different virulence potential. For this reason, we preliminarily characterised via Whole-Genome Sequencing (WGS) some Lm strains for their key genomic features and virulence-associated determinants, assigning the clonal complex (CC). Moreover, the ability of the same strains to adhere to and invade human colon carcinoma cell line Caco-2, evaluating the possible correspondence with their genetic virulence profile, was also assessed. The clinical strains typed belonged to clonal complex (CC)1, CC31, and CC101 and showed a very low invasiveness. The Lm strains isolated from food were assigned to CC1, CC7, CC9, and CC121. All CC1 carried the hypervirulence pathogenicity island LIPI-3 in addition to LIPI-1. Premature stop codons in the inlA gene were found only in Lm of food origin belonging to CC9 and CC121. The presence of LIPI2_inlII was observed in all the CCs except CC1. The CC7 strain, belonging to an epidemic cluster, also carried the internalin genes inlG and inlL and showed the highest level of invasion. In contrast, the human CC31 strain lacked the lapB and vip genes and presented the lowest level of invasiveness. In Lm, the genetic determinants of hypo- or hypervirulence are not necessarily predictive of a cell adhesion and/or invasion ability in vitro. Moreover, since listeriosis results from the interplay between host and virulence features of the pathogen, even hypovirulent clones are able to cause infection in immunocompromised people.

Metabolic Pathway of Hereditary Cancer Disease from PPI-network of DEGS Detected using Mean-of-Mean Method

Uncontrolled growth of cells often results Cancer disease in human body. When it is eventually transmitted throughout the generations in a family, it is referred to as hereditary diseases. Metabolic pathway explains several chemical reactions occurred for growth of a disease and KEGG pathway analysis identifies key genes involved with that disease. At first, differentially expressed genes (DEGs) are detected from cancer gene microarray time series datasets using a new and simple method, Mean of Mean (MoM). The MoM concept is developed from Gregor Johann Mendel's First Law of Heredity or Segregation rule of heredity. Highly expressed (HG) and lowly expressed (LG) genes in two different groups of microarray dataset are identified first using MoM. Then DEGs are found by implementing intersection operation between HG and LG genes. Performance of MoM method is analyzed by Support Vector Machine classifiers (SVMs) on some binary class cancer microarray data samples. Then all results are compared with the performance of other statistical parametric and nonparametric hypothetic tests. It is noticed that performance ofMoMis better than other statistical methods in almost all data sets. Finally, Protein-Protein Interaction Networks (PPINs) are constructed within identified DEGs using web based tool. Lastly, KEGG pathway analysis is performed for all proteins involved in PPINs to obtain list of key genes for growth of cancer disease.

Polymorphism, Allelic and Genotypic Frequencies of κ-Casein and β-LG genes in Egyptian Buffaloes

With a view to detecting the genotypes of both &amp;kappa;-CN and &amp;beta;-LG genes in native populations of Egyptian buffalo using PCR-RFLP technique, 80 randomly, individuals were selected from five geographical locations of some Egyptian provinces. Also, to estimate the population genetic parameters such as, allelic and genotypic frequencies, heterozygosity, and inbreeding coefficient (FIS) of these studied genes. For genotyping, 453 bp PCR product of &amp;kappa;-CN was digested with AcuI and HpyCH4IV (Isoschizomer for MaeII) restriction enzymes while the 247 bp PCR product of &amp;beta;-LG was digested with HaeIII restriction enzyme. PCR-RFLP results discovered polymorphism at the level of &amp;kappa;-CN gene in all studied Egyptian buffaloes with two distinct alleles &amp;ldquo;A&amp;rdquo; and &amp;ldquo;B&amp;rdquo;. PCR-RFLP analysis for &amp;kappa;-CN gene using both restriction enzymes successfully detected that polymorphic status of the studied populations. We recommended using AcuI enzyme which was more capable for differentiating between homozygous (17%) and heterozygous (83%) individuals than HpyCH4IV enzyme which defined only 4% of homozygous individuals and the remaining was heterozygous (96%) individuals. Existence of heterozygosity excess in all studied populations referred to higher degree of genetic variability between individuals within these populations. On contrary, results of PCR-RFLP at the level of &amp;beta;-LG gene revealed a monomorphic pattern of Egyptian buffaloes and genotyped as &amp;ldquo;AA&amp;rdquo; animals which signified that PCR-RFLP assay with HaeIII enzyme for &amp;beta;-LG gene failed to discover any evidence of polymorphism in Egyptian buffalo under the circumstances of this study or all studied animals possess only one allele.

Genotyping of -Lactoglobulin (-Lg) gene by PCR-RFLP in indigenous cattle of Assam, India

The study was conducted to investigate the polymorphism in ?-Lactoglobulin (?-Lg) gene in indigenous cattle of Assam. Genomic DNA from 53 indigenous cattle was extracted and used to study the polymorphism in ?-Lg gene using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) technique. Genetic improvement by selective breeding provides enormous potential to enhance the performance of animals. But, it is a time-consuming exercise as generation interval of cattle is longer. Besides, milk production being a sex limited trait, it is not possible to select male animal on the basis of its own performance. To minimize these problems, selection based on markers can play an important role to carry out genetic improvement of animals before expressing actual target traits. A PCR product of 262 bp obtained upon amplification was subsequently digested with restriction endonuclease BSuRI and yielded three types of restriction pattern, two fragments (153 and 109 bp) for AA genotype, three fragments (109, 79 and 74 bp) for BB genotype and, four fragments (153, 109, 79 and 74 bp) for AB genotype. The frequencies of alleles A and B were found to be 0.2547 and 0.7453 and those of AA, AB and BB genotypes were 0.0943, 0.3207 and 0.5849, respectively. From the present study it could be concluded that the ?-Lg B variant was predominant in the indigenous cattle with the highest frequency of BB homozygote followed by AB heterozygote (BB&gt;AB&gt;AA) and Chi-square (x2) test revealed that the population under study was in Hardy-Weinberg Equilibrium.

Transcriptome and MassARRAY analysis for identification of transcripts and SNPs for growth traits of the swimming crab Portunus trituberculatus

Portunus trituberculatus is an important farmed crustacean in China. Growth traits are important economic traits that are controlled by many genes. The aim of this study was to screen growth-related genes using Illumina paired-end sequencing technology, and to identify growth-related SNPs in P. trituberculatus by association analysis. A total of 36,599,712 and 29,247,160 qualified Illumina read pairs for small size (SG) and large size crabs (LG) were obtained, giving rise to total clean bases of 7.14 and 5.72G, respectively. A gene expression comparison showed that a total of 117 unigenes were differentially expressed between SG and LG, including 49 up-regulated and 68 down-regulated unigenes in SG and LG, respectively. GO functional enrichment analysis results showed up-regulated genes in LG were involved in three biological processes. These were cell communication, ovarian follicle cell development and cell division. Down-regulated genes in LG were involved in five biological processes, which were chitin metabolism, carbohydrate metabolism, ATP hydrolysis coupled proton transport, the steroid hormone mediated signaling pathway and fatty acid biosynthesis. Association analysis identified one growth-associated SNP, which was located in hemocyanin (comp58070-R31). This research has provided information on key genes, SNPs and biological processes related to growth, which will increase the efficiency of selective breeding programs for this important species.

Nucleotide sequencing and DNA polymorphism studies of beta-lactoglobulin gene in native Saudi goat breeds in relation to milk yield

β-Lactoglobulin (β-LG) is the dominant non-casein whey protein found in milk of bovine and of most ruminants. The amino acid sequence of β-LG along with its 3-dimensional structure illustrates linkage with the lipocalin superfamily. Preliminary studies in goats indicated that milk yield can be influenced by poly - morphism in genes coding for whey proteins. The aim of this study is to identify and evaluate the incidence of functional polymorphisms in the exonic and intronic portions of β- LG gene in native Saudi goat breeds (Ardi, Habsi and Harri). Blood samples were collected from 300 animals (100 for each breed) and genomic DNA was extracted using QIAamp DNA extraction kit. A fragment of the β- LG gene from exon 7 to 3' flanking region

Polymorphism of β-lacto globulin gene in Barki sheep breed

?-LG gene polymorphism at the DNA level has been analyzed by PCR-RFLP and reported two genetic variants of the ? -LG gene in Pag native and Leccese sheep breeds. However, no study has been reported in Barki Egyptian local sheep breed regarding ? -LG polymorphism at DNA level. The association of ?- LG polymorphism with milk production and manufacturing properties of the cheese in sheep have been extensively studied. However, the results are conflicting, indicating either superiority of a given ? - LG variant or absence of relationship. The fingerprinting of ? -LG gene and this relation has not clarified yet in local Barki and other sheep breeds raised in Egypt. Regarding the importance of Barki sheep breed as a local adaptive breed in Egypt, this preliminary study aimed to fingerprint ?-LG gene in the Barki sheep breed, in an attempt to have clear image about genotypes of ?-LG gene in this breed. A total of 60 adult females Barki ewes were fingerprinted for ?-LG gene by the PCR-RFLP method. The sheep are raised by farmers in North West coast (Borg EL-Arab, Alexandria, Egypt) in which sheep are living in natural habitats. The amplified product was observed as 120 bp and the restriction digestion with Rsa I revealed three genotypes, namely AA, and BB at the ?-LG locus. The frequencies of AA, and BB genotypes were 0.45, 0.30, 0.25, respectively.

Polymorphism of β-lacto globulin gene in Barki and Damascus and their cross bred goats in relation to milk yield

?-LG gene polymorphism at the DNA level has been analyzed by PCR-RFLP and reported two novel genetic variants of the ?-LG gene in Indian goats. However, no study has been reported in Barki Egyptian local goat breed, Damascus and their crossbred goats regarding ?-LG polymorphism at DNA level. The association of ?-LG polymorphism with milk yield has been reported in cows, sheep and Indian goats. However, this relation has not clarified yet in local or foreign goat breeds raised in Egypt. This study aimed to fingerprint ?-LG gene in the Barki, Damascus and their crossbred goats in an attempt to have clear image about genotypes of ?-LG gene in these breeds in relation to their milk yield. A total of 120 adult female does belonging to these three different genetic groups were analyzed by the PCR-RFLP method. The goats are raised by farmers in North West coast (Borg EL-Arab, Alexandria, Egypt) in which goats are living in natural habitats. The amplified product was observed as 426 bp and the restriction digestion with SacII revealed three genotypes, namely AA, AB and BB at the ?-LG locus. The frequencies of AA, AB and BB genotypes were 0.1, 0.8, 0.1; 0.85, 0.1, 0.05; 0.41, 0.51, 0.08 in the Barki and Damascus and their crossbred goats, respectively. The frequency of (AA)genotype was higher in Damascus breed than Barki and Damascus x Barki crossbred and according to reference milk production was associated with significantly higher milk production in this goat breed as compared by the two other goat breeds.

Hybrid necrosis genes in aboriginal wheats of Middle Asia in the light of the problem of the primary centers of biodiversity of the Triticum L. genus

The distribution of hybrid necrosis genes in aboriginal wheats of Middle Asia (Tadzhikistan, Kyrghyzstan, Uzbekistan, and Turkmenistan) was studied. Necrotic genotypes were identified in 154 samples including 147 samples of Triticum aestivum, six samples of T. compactum, and one T. antiquorum sample. The absence of the Ne2 gene in this region was shown. About 43.5% of the samples examined were of the Ne1ne2 genotype, while 56.5% of samples had the genotype ne1ne2. The frequency of the Ne1 gene in T. aestivum from Uzbekistan and Turkmenistan was higher than in Tadzhikistan and Kyrghyzstan (χ2 = 13.9; d.f. = 1). The height above sea level had no impact on Ne1 frequencies in cultivated wheats. No correlation between the frequencies of Ne1 and lg genes was revealed. It was suggested that the Middle Asia is a secondary route of wheat distribution from its primary center of origin.

Nucleosome organisation of the beta-lactoglobulin gene. Transcription complex formation.

In most mammals, the major whey protein beta-lactoglobulin (beta lg) represents a marker for tissue-specific, temporally regulated gene expression in the mammary gland. Prolactin, the major lactogenic stimulus which activates beta lg expression acts through a cytoplasmic signalling cascade ending in the activation of the transcription factor STAT5. Although much is known about the complexities of this signalling cascade, little is known about how this transcription factor functions within the context of chromatin. Using DNaseI as a probe of chromatin structure we have identified temporally regulated elements within the ovine beta lg gene domain. The appearance of these hypersensitive sites accompanies changes in expression state of the beta lg gene. Changes in DNaseI hypersensitivity at the proximal promoter region, while reflecting STAT5 activation, is not dependent upon STAT5 interaction at this site. We have mapped the nucleosome positions over the entire beta lg gene, both in vitro using the monomer extension assay and in vivo using cuprous phenanthroline to probe for nucleosome-linker positions. The specific positioning pattern detected, which reflects strong sequence-directed positioning over the proximal promoter, complement the STAT5 consensus sites within this region. The comparison of both the functional and chromatin data enables a model for beta lg gene transcription to be developed.

The gene cluster inlC2DE of Listeria monocytogenes contains additional new internalin genes and is important for virulence in mice.

In this work we identified and characterized a gene cluster containing three internalin genes of Listeria monocytogenes EGD. These genes, termed inlG, inlH and inlE, encode proteins of 490, 548 and 499 amino acids, respectively, which belong to the family of large, cell wall-bound internalins. The inlGHE gene cluster is flanked by two listerial house-keeping genes encoding proteins homologous to the 6-phospho-beta-glucosidase and the succinyl-diaminopimelate desuccinylase of E. coli. A similar internalin gene cluster, inlC2DE, localised to the same position on the L. monocytogenes EGD chromosome was recently described in a different isolate (Dramsi S, Dehoux P, Lebrun M, Goossens PL, Cossart P (1997) Infect Immun 65: 1615-1625). Sequence comparison of the two inl gene clusters indicates that inlG is a new internalin gene, while inlH was generated by a site-specific recombination, leading to an in-frame deletion which removed the 3'-terminal end of inlC2 and the 5'-terminal part of inlD. The third gene of the inlGHE cluster, inlE, is almost identical to the previously reported inlE gene. Our data show that the inlGHE gene cluster is probably transcribed from a major PrfA-independent promoter located upstream of inlG. PCR analysis revealed the presence of the newly identified inl genes inlG and inlH in most L. monocytogenes isolates tested. A mutant which has lost inlG, inlH and inlE by an in-frame deletion exhibited, after oral infection of mice, a significant loss in virulence and shows drastically reduced numbers of viable bacteria in both liver and spleen when compared to the wild-type strain.

Immunoglobulin transgenes as targets for somatic hypermutation.

This review describes studies on somatic hypermutation of immunoglobulin genes that were started in the mid-80s in collaboration with Ralph Brinster. Almost all of the experiments were carried out using Ig transgenes as targets for the somatic mutation mechanism. Ig transgenes can be very good targets of somatic mutation, despite many different transgene integration sites. Thus, the required cis-acting elements must be present within the approximately 10 kb of the transgene. Only the Ig variable region and its proximate flanks are mutated, not the constant region in unmanipulated sequences. Several Ig gene enhancers are permissive for somatic mutation and they do not have to be associated with the Ig promoter they normally interact with. However, the mutation process does seem to be specific for Ig genes. No mutations were found in several housekeeping genes isolated from cells that had very high levels of somatic hypermutation of their Ig genes. This suggests that the Ig enhancers provide the lg gene specificity. An exception is the Bcl-6 gene, encoding a transcription factor, which was found to be mutated in normal human memory B cells. When the transcriptional promoter that is located upstream of the variable region is duplicated upstream of the constant region, this region is mutated as well. This suggests a transcription coupled model in which a mutator factor associates with the RNA polymerase at the initiation of transcription, travels with the polymerase during elongation, and causes mutations during polymerase pausing. Our recent data with an artificial substrate for somatic mutation suggest that the mutations are increased by increased stability of the secondary structures in the nascent RNA, and the specific nucleotides that are mutated are due to preferences of a mutator factor.

Molecular genetics and lymphoproliferative disorders.

Clonality of T- and B-cell lymphoproliferative disorders can be determined by gene rearrangement studies when morphology and surface immunostaining are nondiagnostic. TcR and lg gene rearrangements have been demonstrated in many different hematologic disorders and TcR gene rearrangement has been particularly useful in the diagnosis of patients with CD8 large granular lymphocyte leukemias. TcR gene rearrangement may also be useful to distinguish Hodgkin's disease from T-cell non-Hodgkin's lymphoma. Gene rearrangement is usually performed by Southern analysis, and it is beneficial to run multiple enzyme-probe combinations to maximize the detection of clonal rearrangements. More recently, several laboratories have begun to use polymerase chain reaction (PCR) for gene rearrangement analysis. PCR offers an improved turnaround time, eliminates partial digestion artifacts, and allows for the use of paraffin embedded material. In addition to rearrangements of the TcR and lg genes, analysis of alterations in other genes such as bcl-1, bcl-2, bcl-6, and c-myc are also useful as clonal markers and aid in the classification of lymphomas.

Characterization of human CD7 transgenic mice.

CD7 is a 40-kDa transmembrane glycoprotein member of the lg gene superfamily expressed on most peripheral blood T lymphocytes and NK cells. CD7 is also expressed on myeloid, NK, B, and T cell precursors during adult hematopoiesis. Because Thy-1 is absent in human thymocytes and peripheral blood T cells and shows structural similarities to the human CD7 gene, we have suggested that human CD7 may be a functional homologue in humans of mouse Thy-1. To study the tissue-specific expression of the CD7 gene utilizing its own promoter, we constructed transgenic mice that contained both the coding and flanking regions of the human CD7 gene. We found that human CD7 was expressed in transgenic mice in T, B, NK, and myeloid lineages and was induced with T cell activation. Unlike the expression of CD7 in humans, the CD7 transgene was present on mature B lymphocytes and macrophages. Like mouse Thy-1, transgenic human CD7 was expressed in immature and mature T cells and in Sca-1+ bone marrow mononuclear cells. Unlike mouse Thy-1, the human CD7 transgene was not expressed in mouse brain or fibroblasts. The human CD7 transgene was expressed during fetal development before mouse Thy-1 in fetal liver mononuclear cells. Expression of the human CD7 transgene did not alter mouse thymopiesis or Thy-1 expression. Taken together, these data demonstrated that the CD7 transgene contained sufficient regulatory regions to direct hematopoietic expression and mitogenic induction. The pattern of CD7 transgene expression more closely resembled that of CD7 in humans than that of mouse Thy-1.

Mutations of the chloramphenicol acetyl transferase transgene driven by the immunoglobulin promoter and intron enhancer.

Transgenic mice containing the chloramphenicol acetyl transferase (CAT) gene driven by the immunoglobulin (Ig) VH promoter and the heavy chain intron enhancer were prepared to increase our understanding of the mechanism responsible for somatic mutation. The transgene showed similar tissue specificity in terms of expression as endogenous Ig genes. Hybridomas were prepared after multiple immunization of a transgenic mouse with (4-hydroxy-3-nitrophenyl)acetyl-chicken gammaglobulin, a thymus-dependent antigen. We analyzed mutations in the CAT coding region as well as in the region 5' upstream of the promoter after amplification of DNA using the PCR followed by sequencing of cloned DNA. Mutation detection enhancement gel electrophoresis was also used to detect mutations. Only a single band was observed in PCR products from the region 5' upstream of the promoter and from the enhancer, whereas in those from the CAT coding region, three out of 11 hybridomas showed multiple bands. In DNA sequences of the CAT coding region from these three hybridomas, a total of six nucleotide substitutions were observed, but none in the region 5' to the promoter. The results of DNA sequencing and the electrophoresis were in good agreement, suggesting that mutation occurred only in the CAT gene but not in the region 5' to the promoter or the enhancer. The rate of mutation in the CAT gene was estimated to be 1.6 x 10(-5) per base pair per cell division, which was a lower limit of the rate for somatic mutation reported for Ig genes. Location and distribution of mutations were similar to that of the Ig gene, suggesting that (i) mutation in the CAT gene was induced by a mechanism similar to that occurring in the Ig gene and (ii) the mutator mechanism can operate in a non-Ig gene provided it is flanked by the VH promoter and heavy chain intron enhancer.

Response assessment in chronic lymphocytic leukemia after fludarabine plus prednisone: clinical, pathologic, immunophenotypic, and molecular analysis [see comments

The goals of this study were to evaluate the response to treatment in chronic lymphocytic leukemia (CLL) according to clinical, pathologic, immunophenotypic, and molecular features, as well as to address the clinical significance of each finding. One hundred fifty-nine CLL patients with either advanced Rai stage III or IV (81 patients) or progressive Rai stage 0 to II (78 patients) were treated with fludarabine (30 mg/m2/d intravenously every day for 5 days) plus prednisone (30 mg/m2/d orally daily for 5 days). Thirty-six patients were previously untreated. The response rates were 12% complete response (CR), 30% nodular complete response (nCR), and 18% partial response (PR). In all patients who achieved a complete response (both CR and nCR) less than 30% of nucleated cells were lymphocytes on marrow aspirate differential analysis; however, nCR patients had residual nodular and/or interstitial lymphocyte involvement on marrow biopsy examination. There was no evidence of leukemic infiltration on marrow biopsy examination in CR patients. With a median follow-up of 35 months, comparison of time to progression in the CR and nCR groups at 2 years showed a projected 87% versus 55% progression-free survival (P less than .03). Residual disease assessment by flow cytometry using simultaneous dual-color staining on blood and marrow lymphocytes was also performed on each patient. Residual disease was determined by the expression of CD5 on B lymphocytes and the monoclonality of surface light-chain expression. After six courses of fludarabine plus prednisone, no residual disease was detected by flow cytometry in 89% of the CRs, 51% of the nCRs, and 19% of the PRs. Clinical residual disease in PR patients with no residual disease detectable by flow cytometry was limited to lymph-adenopathy. Time to progression at 2 years was longer in CR and nCR patients having no residual disease detected by flow cytometry (84% v 39% 2-year progression-free survival, P less than .001). Posttreatment lg gene rearrangement analysis using JH, J kappa, and C lambda probes demonstrated no rearranged bands and a return to the germline configuration in five of seven CRs and two of eight nCRs studied. The molecular studies were concordant with the dual-parameter immunophenotype results and none of the patients who reverted to a germline DNA pattern after treatment have experienced relapse. The absence of detectable minimal residual disease by bone marrow biopsy, dual-color flow cytometry, and lg gene rearrangement analysis is achieveable in CLL with fludarabine and is predictive of the response duration.

Response Assessment in Chronic Lymphocytic Leukemia After Fludarabine Plus Prednisone: Clinical, Pathologic, Immunophenotypic, and Molecular Analysis

Abstract The goals of this study were to evaluate the response to treatment in chronic lymphocytic leukemia (CLL) according to clinical, pathologic, immunophenotypic, and molecular features, as well as to address the clinical significance of each finding. One hundred fifty-nine CLL patients with either advanced Rai stage III or IV (81 patients) or progressive Rai stage 0 to II (78 patients) were treated with fludarabine (30 mg/m2/d intravenously every day for 5 days) plus prednisone (30 mg/m2/d orally daily for 5 days). Thirty-six patients were previously untreated. The response rates were 12% complete response (CR), 30% nodular complete response (nCR), and 18% partial response (PR). In all patients who achieved a complete response (both CR and nCR) less than 30% of nucleated cells were lymphocytes on marrow aspirate differential analysis; however, nCR patients had residual nodular and/or interstitial lymphocyte involvement on marrow biopsy examination. There was no evidence of leukemic infiltration on marrow biopsy examination in CR patients. With a median follow-up of 35 months, comparison of time to progression in the CR and nCR groups at 2 years showed a projected 87% versus 55% progression-free survival (P less than .03). Residual disease assessment by flow cytometry using simultaneous dual-color staining on blood and marrow lymphocytes was also performed on each patient. Residual disease was determined by the expression of CD5 on B lymphocytes and the monoclonality of surface light-chain expression. After six courses of fludarabine plus prednisone, no residual disease was detected by flow cytometry in 89% of the CRs, 51% of the nCRs, and 19% of the PRs. Clinical residual disease in PR patients with no residual disease detectable by flow cytometry was limited to lymph-adenopathy. Time to progression at 2 years was longer in CR and nCR patients having no residual disease detected by flow cytometry (84% v 39% 2-year progression-free survival, P less than .001). Posttreatment lg gene rearrangement analysis using JH, J kappa, and C lambda probes demonstrated no rearranged bands and a return to the germline configuration in five of seven CRs and two of eight nCRs studied. The molecular studies were concordant with the dual- parameter immunophenotype results and none of the patients who reverted to a germline DNA pattern after treatment have experienced relapse. The absence of detectable minimal residual disease by bone marrow biopsy, dual-color flow cytometry, and lg gene rearrangement analysis is achieveable in CLL with fludarabine and is predictive of the response duration.

A second ferritin L subunit is encoded by an intronless gene in the mouse.

Multiple homologous sequences for the ferritin L subunit are present in mammalian genomes, but so far, only one expressed gene has been described. Here we report the isolation of a cDNA from a mouse bone marrow library, corresponding to an isoform of the mouse ferritin L subunit. This new subunit, that we named Lg, differs from the L subunit of ten amino acids. Specific amplification of mouse genomic DNA using the polymerase chain reaction (PCR) confirmed the presence of this Lg sequence in the mouse genome but also suggested that it must be encoded by an intronless gene. Using a series of different Lg-specific oligonucleotides as probes, we subsequently isolated a genomic clone containing an uninterrupted sequence, identical to the Lg cDNA. This Lg gene lacks introns and does not contain the 28 base pairs (bp) conserved motif usually present at the 5' end of most ferritin mRNAs, which confers translational regulation by iron. When transiently transfected into K562 cells, this Lg genomic clone is actively transcribed, suggesting that, although it possesses the characteristics of a processed pseudogene, it is likely to correspond to the gene encoding this new ferritin subunit.

Differentiation in B-Precursor Acute Lymphoblastic Leukemia Cell Populations With CD34-Positive Subpopulations

Abstract B-precursor acute lymphoblastic leukemia bone marrow specimens that contained subpopulations of cells with immunophenotypes corresponding to early (CD34) and late (CD20) and (CD22) stages of normal B-cell differentiation were studied. Subpopulations of cells were isolated according to immunophenotype and then analyzed by both a clonogenic assay and molecular genetic methods. Clonal equivalence of the early and late immunophenotypic subpopulations was confirmed for each case by the demonstration of identical lg gene rearrangements. The in vitro colony-forming assay consistently showed a growth advantage for the CD34+ subpopulations over the CD34- subpopulations. CD34 mRNA was detected readily in these isolated precursor cells. When two specimens in which virtually all of the leukemia cells were CD34+ and CD34+CD20+ and CD34+CD22+ subpopulations were also present the CD34 mRNA was limited to the cells without the late-stage differentiation antigens on their surface. Furthermore, the c-myb mRNA was found only in the subpopulations that also contained CD34 mRNA. Our results show that a limited program of differentiation reminiscent of normal B-cell development may be present in this leukemia.