Csl Gene Research Articles

Genome-wide identification and functional analysis of the cellulose synthase-like gene superfamily in common oat (Avena sativa L.)

Hemicelluloses constitute approximately one-third of the plant cell wall and can be used as a dietary fiber and food additive, and as raw materials for biofuels. Although genes involved in hemicelluloses synthesis have been investigated in some model plants, no comprehensive analysis has been conducted in common oat at present. In this study, we identified and systematically analyzed the cellulose synthase-like gene (Csl) family members in common oat and investigated them using various bioinformatics tools. The results showed that there are 76 members of the oat Csl gene family distributed on 17 chromosomes, and phylogenetic analysis indicated that the 76 Csl genes belong to the CslA, CslC, CslD, CslE, CslF, CslH, and CslJ subfamilies. A total of 14 classes of cis-acting elements were identified in the promoter regions, including hormone response, light response, cell development, and defense stress elements. The collinearity analysis identified 28 pairs of segmentally duplicated genes, most of which were found on chromosomes 2D and 6A. Expression pattern analysis showed that oat Csl genes display strong tissue-specific expression; of the 76 Csl genes, 33 were significantly up-regulated in stems and 30 were up-regulated in immature seeds. The expression of most members of the AsCsl gene family is repressed by abiotic stress, while the expression of some members is up-regulated by light. Immunoelectron microscopy shows that the product of AsCsl61, a member of CslF subfamily, mediates (1,3; 1,4)-β-D-glucan synthesis in transgenic Arabidopsis. These findings provide a fundamental understanding of the structural, functional, and evolutionary features of the oat Csl genes and may contribute to our general understanding of hemicellulose biosynthesis. Moreover, this information will be helpful in designing experiments for genetic manipulation of mixed-linkage glucan (MLG) synthesis with the goal of quality improvement in oat.

Genome-wide identification and functional analysis of Cellulose synthase gene superfamily in Fragaria vesca.

The Cellulose synthase (CesA) and Cellulose synthase-like (Csl) gene superfamilies encode key enzymes involved in the synthesis of cellulose and hemicellulose, which are major components of plant cell walls, and play important roles in the regulation of fruit ripening. However, genome-wide identification and functional analysis of the CesA and Csl gene families in strawberry remain limited. In this study, eight CesA genes and 25 Csl genes were identified in the genome of diploid woodland strawberry (Fragaria vesca). The protein structures, evolutionary relationships, and cis-acting elements of the promoter for each gene were investigated. Transcriptome analysis and quantitative real-time PCR (qRT-PCR) results showed that the transcript levels of many FveCesA and FveCsl genes were significantly decreased during fruit ripening. Moreover, based on the transcriptome analysis, we found that the expression levels of many FveCesA/Csl genes were changed after nordihydroguaiaretic acid (NDGA) treatment. Transient overexpression of FveCesA4 in immature strawberry fruit increased fruit firmness and reduced fresh fruit weight, thereby delaying ripening. In contrast, transient expression of FveCesA4-RNAi resulted in the opposite phenotypes. These findings provide fundamental information on strawberry CesA and Csl genes and may contribute to the elucidation of the molecular mechanism by which FveCesA/Csl-mediated cell wall synthesis regulates fruit ripening. In addition, these results may be useful in strawberry breeding programs focused on the development of new cultivars with increased fruit shelf-life.

Genome-wide bioinformatics analysis of Cellulose Synthase gene family in common bean (Phaseolus vulgaris L.) and the expression in the pod development

BackgroundCesA and Csl gene families, which belong to the cellulose synthase gene superfamily, plays an important role in the biosynthesis of the plant cell wall. Although researchers have investigated this gene superfamily in several model plants, to date, no comprehensive analysis has been conducted in the common bean.ResultsIn this study, we identified 39 putative cellulose synthase genes from the common bean genome sequence. Then, we performed a bioinformatics analysis of this gene family involving sequence alignment, phylogenetic analysis, gene structure, collinearity analysis and chromosome location. We found all members possess a cellulose_synt domain. Phylogenetic analysis revealed that these cellulose synthase genes may be classified into five subfamilies, and that members in the same subfamily share conserved exon-intron distribution and motif compositions. Abundant and distinct cis-acting elements in the 2 k basepairs upstream regulatory regions indicate that the cellulose synthase gene family may plays a vital role in the growth and development of common bean. Moreover, the 39 cellulose synthase genes are distributed on 10 of the 11 chromosomes. Additionally expression analysis shows that all CesA/Csl genes selected are constitutively expressed in the pod development.ConclusionsThis research reveals both the putative biochemical and physiological functions of cellulose synthase genes in common bean and implies the importance of studying non-model plants to understand the breadth and diversity of cellulose synthase genes.

Genome-Wide Identification of Banana Csl Gene Family and Their Different Responses to Low Temperature between Chilling-Sensitive and Tolerant Cultivars.

The cell wall plays an important role in responses to various stresses. The cellulose synthase-like gene (Csl) family has been reported to be involved in the biosynthesis of the hemicellulose backbone. However, little information is available on their involvement in plant tolerance to low-temperature (LT) stress. In this study, a total of 42 Csls were identified in Musa acuminata and clustered into six subfamilies (CslA, CslC, CslD, CslE, CslG, and CslH) according to phylogenetic relationships. The genomic features of MaCsl genes were characterized to identify gene structures, conserved motifs and the distribution among chromosomes. A phylogenetic tree was constructed to show the diversity in these genes. Different changes in hemicellulose content between chilling-tolerant and chilling-sensitive banana cultivars under LT were observed, suggesting that certain types of hemicellulose are involved in LT stress tolerance in banana. Thus, the expression patterns of MaCsl genes in both cultivars after LT treatment were investigated by RNA sequencing (RNA-Seq) technique followed by quantitative real-time PCR (qPCR) validation. The results indicated that MaCslA4/12, MaCslD4 and MaCslE2 are promising candidates determining the chilling tolerance of banana. Our results provide the first genome-wide characterization of the MaCsls in banana, and open the door for further functional studies.

Synthetic lethality across normal tissues is strongly associated with cancer risk, onset, and tumor suppressor specificity.

Various characteristics of cancers exhibit tissue specificity, including lifetime cancer risk, onset age, and cancer driver genes. Previously, the large variation in cancer risk across human tissues was found to strongly correlate with the number of stem cell divisions and abnormal DNA methylation levels. Here, we study the role of synthetic lethality in cancer risk. Analyzing normal tissue transcriptomics data in the Genotype-Tissue Expression project, we quantify the extent of co-inactivation of cancer synthetic lethal (cSL) gene pairs and find that normal tissues with more down-regulated cSL gene pairs have lower and delayed cancer risk. Consistently, more cSL gene pairs become up-regulated in cells treated by carcinogens and throughout premalignant stages in vivo. We also show that the tissue specificity of numerous tumor suppressor genes is associated with the expression of their cSL partner genes across normal tissues. Overall, our findings support the possible role of synthetic lethality in tumorigenesis.

Abstract 36: Synthetic lethality across normal tissues is strongly associated with cancer risk, onset, and tumor suppressor specificity

Abstract The tissue-specificity of cancer and cancer risk is a fundamental open research question. Beyond advancing our understanding of carcinogenesis, elucidating the factors underlying cancer risk may also contribute to cancer prevention. Recent studies have shown that the variation in tissue cancer risk can be explained by the number of tissue stem cell divisions occurring during the lifetime and by abnormal levels of DNA methylation. While cancer risk is likely not determined by a single factor, no other factor has been reported since to account for this fundamental variation. Here we show that cancer synthetic lethality (SL) is another strong contributor of cancer risk in human tissues. SL is a well-known type of genetic interaction where cell death occurs under the combined inactivation of two paired SL genes but not either of them alone. Targeting SLs has been recognized as a highly valuable approach for cancer treatment. We hypothesized that since down-regulated cancer SL (cSL) gene pairs reduce the viability of cancer cells, they may impede the malignant transformation of normal cells, thus modulating cancer risk. Utilizing several recently published large-scale cancer SL networks, we systematically quantified the cancer SL load (defined as the fraction of down-regulated cancer SL gene pairs) in numerous normal and cancer tissues from the TCGA and GTEx datasets. Our key findings are: 1. The cSL loads are lower in cancers vs their matched normal tissues. Furthermore, we find that the cSL load decreases progressively as cancers develop from normal tissues through multiple stages of pre-malignant lesions. These results testify that high cSL load is detrimental to cancer cells, acting as a barrier to cancer development. That is, as normal cells undergo malignant transformation, they need to reactivate at least some of the down-regulated cSL genes for the emerging cancer cells to survive. 2. In accordance with these observations, we find that cSL load in normal tissues is strongly inversely correlated with their lifetime cancer risk. 3. We find that cSL load is the first identified predictor of cancer onset time across different normal tissues - higher cSL load in the younger population is associated with later onset of cancers in that tissue. 4. cSL are important contributors of the tissue/cancer-type specificity of numerous tumor suppressor genes (like BRCA1) - that is, the activity state of cSL partners of quite a few tumor suppressor genes predicts the specific tissues in which they are known to drive cancer. Taken together, these results show that synthetic lethality load in normal tissues is a novel important biological contributor of cancer risk in humans. While synthetic lethality has been attracting tremendous attention as a way to identify cancer vulnerabilities and target them, this is the first time that its role in mediating cancer development is uncovered. Citation Format: Nishanth Ulhas Nair, Kuoyuan Cheng, Joo Sang Lee, Eytan Ruppin. Synthetic lethality across normal tissues is strongly associated with cancer risk, onset, and tumor suppressor specificity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 36.

A genome-wide analysis of the cellulose synthase-like (Csl) gene family in maize

Cell walls play an important role in the structure and morphology of plants as well as in responses to various biotic and abiotic stresses. Although the comprehensive analysis of genes involved in cellulose synthase has been performed in model plants, such as Arabidopsis thaliana and rice, information regarding cellulose synthase-like (Csl) genes in maize is limited. In this study, a total of 56 members of Csl gene family were identified in maize genome and classified into six subfamilies. Analysis of gene structure and conserved motif indicated functional similarities among the ZmCsl proteins within the same subfamily. Additionally, the 56 ZmCsl genes were dispersed on 10 chromosomes. The expression patterns of ZmCsl genes in different tissues using the transcriptome data revealed that most of ZmCsl genes had a relatively high expression in root and tassel tissues. Moreover, the expression profiles of ZmCsl genes under drought and re-watering indicated that the expression of ZmCsl genes were mainly responsive to early stage of drought stress. The protein-protein interaction network of ZmCsl proposed some potentially interacting proteins. The data presented a comprehensive survey of Csl gene family in maize. The detailed description of maize Csl genes will be beneficial to understand their structural, functional, and evolutionary features and provide an important foundation for studying the roles of ZmCsl genes in response to biotic and abiotic stresses.

Molecular cloning and functional characterization of a homolog of the transcriptional regulator CSL in Litopenaeus vannamei

The Notch signaling pathway transcriptional regulator, CSL (also called as CBF1, Suppressor of Hairless or Lag-1 in different species, generally designated as CSL1), is not only associated with cell proliferation and differentiation but also involved in tumorigenesis, inflammation and immune regulation in vertebrates. We recently showed that Notch signaling was involved in the immune response of Litopenaeus vannamei shrimp. However, as an important transcriptional regulator of this pathway, whether or not shrimp CSL was also involved in immune response had not been explored. Here, we cloned and characterized the CSL gene in L. vannamei (LvCSL), which has a 2271 bp open reading frame (ORF) encoding a putative protein of 756 amino acids, and contains two conserved Lag1-DNA bind as well as beta trefoil domains (BTD). LvCSL clustered with invertebrates in the phylogenetic tree and closely related to the RBP Jk X1 of Parasteatoda tepidariorum. The transcript level of LvCSL analyzed by quantitative polymerase chain reaction (qPCR) showed that LvCSL was widely expressed in all tissues tested, with induced levels observed in the hepatopancreas and hemocytes following immune challenge with Vibrio parahaemolyticus, Streptoccocus iniae, lipopolysaccharide (LPS), and white spot syndrome virus (WSSV), therefore, suggesting LvCSL involvement in shrimp immune response to pathogens. Besides, LvCSL knockdown decreased the expression of proliferation-related genes (LvHey2 and LvAstakine), and attenuated the expression of immune-related genes L. vannamei hypoxia inducible factor alpha (LvHIF-α), LvLectin and L. vannamei small subunit hemocyanin (LvHMCS) in shrimp hemocytes, as well as significantly decreased total hemocyte count. Moreover, high cumulative mortality was observed in LvCSL depleted shrimp challenged with V. parahaemoliticus. In conclusion, our present data strongly suggest that LvCSL is an important factor in shrimp, vital for shrimp survival and contributing to immune resistance to pathogens.

Genome-wide analysis of the cellulose synthase-like (Csl) gene family in bread wheat (Triticum aestivum L.)

BackgroundHemicelluloses are a diverse group of complex, non-cellulosic polysaccharides, which constitute approximately one-third of the plant cell wall and find use as dietary fibres, food additives and raw materials for biofuels. Genes involved in hemicellulose synthesis have not been extensively studied in small grain cereals.ResultsIn efforts to isolate the sequences for the cellulose synthase-like (Csl) gene family from wheat, we identified 108 genes (hereafter referred to as TaCsl). Each gene was represented by two to three homeoalleles, which are named as TaCslXY_ZA, TaCslXY_ZB, or TaCslXY_ZD, where X denotes the Csl subfamily, Y the gene number and Z the wheat chromosome where it is located. A quarter of these genes were predicted to have 2 to 3 splice variants, resulting in a total of 137 putative translated products. Approximately 45% of TaCsl genes were located on chromosomes 2 and 3. Sequences from the subfamilies C and D were interspersed between the dicots and grasses but those from subfamily A clustered within each group of plants. Proximity of the dicot-specific subfamilies B and G, to the grass-specific subfamilies H and J, respectively, points to their common origin. In silico expression analysis in different tissues revealed that most of the genes were expressed ubiquitously and some were tissue-specific. More than half of the genes had introns in phase 0, one-third in phase 2, and a few in phase 1.ConclusionDetailed characterization of the wheat Csl genes has enhanced the understanding of their structural, functional, and evolutionary features. This information will be helpful in designing experiments for genetic manipulation of hemicellulose synthesis with the goal of developing improved cultivars for biofuel production and increased tolerance against various stresses.

Repeat-length variation in a wheat cellulose synthase-like gene is associated with altered tiller number and stem cell wall composition.

The tiller inhibition gene (tin) that reduces tillering in wheat (Triticum aestivum) is also associated with large spikes, increased grain weight, and thick leaves and stems. In this study, comparison of near-isogenic lines (NILs) revealed changes in stem morphology, cell wall composition, and stem strength. Microscopic analysis of stem cross-sections and chemical analysis of stem tissue indicated that cell walls in tin lines were thicker and more lignified than in free-tillering NILs. Increased lignification was associated with stronger stems in tin plants. A candidate gene for tin was identified through map-based cloning and was predicted to encode a cellulose synthase-like (Csl) protein with homology to members of the CslA clade. Dinucleotide repeat-length polymorphism in the 5'UTR region of the Csl gene was associated with tiller number in diverse wheat germplasm and linked to expression differences of Csl transcripts between NILs. We propose that regulation of Csl transcript and/or protein levels affects carbon partitioning throughout the plant, which plays a key role in the tin phenotype.

Genetics and physiology of cell wall polysaccharides in the model C4 grass, Setaria viridis spp.

BackgroundSetaria viridis has emerged as a model species for the larger C4 grasses. Here the cellulose synthase (CesA) superfamily has been defined, with an emphasis on the amounts and distribution of (1,3;1,4)-β-glucan, a cell wall polysaccharide that is characteristic of the grasses and is of considerable value for human health.MethodsOrthologous relationship of the CesA and Poales-specific cellulose synthase-like (Csl) genes among Setaria italica (Si), Sorghum bicolor (Sb), Oryza sativa (Os), Brachypodium distachyon (Bradi) and Hordeum vulgare (Hv) were compared using bioinformatics analysis. Transcription profiling of Csl gene families, which are involved in (1,3;1,4)-β-glucan synthesis, was performed using real-time quantitative PCR (Q-PCR). The amount of (1,3;1,4)-β-glucan was measured using a modified Megazyme assay. The fine structures of the (1,3;1,4)-β-glucan, as denoted by the ratio of cellotriosyl to cellotetraosyl residues (DP3:DP4 ratio) was assessed by chromatography (HPLC and HPAEC-PAD). The distribution and deposition of the MLG was examined using the specific antibody BG-1 and captured using fluorescence and transmission electron microscopy (TEM).ResultsThe cellulose synthase gene superfamily contains 13 CesA and 35 Csl genes in Setaria. Transcript profiling of CslF, CslH and CslJ gene families across a vegetative tissue series indicated that SvCslF6 transcripts were the most abundant relative to all other Csl transcripts. The amounts of (1,3;1,4)-β-glucan in Setaria vegetative tissues ranged from 0.2% to 2.9% w/w with much smaller amounts in developing grain (0.003% to 0.013% w/w). In general, the amount of (1,3;1,4)-β-glucan was greater in younger than in older tissues. The DP3:DP4 ratios varied between tissue types and across developmental stages, and ranged from 2.4 to 3.0:1. The DP3:DP4 ratios in developing grain ranged from 2.5 to 2.8:1. Micrographs revealing the distribution of (1,3;1,4)-β-glucan in walls of different cell types and the data were consistent with the quantitative (1,3;1,4)-β-glucan assays.ConclusionThe characteristics of the cellulose synthase gene superfamily and the accumulation and distribution of (1,3;1,4)-β-glucans in Setaria are similar to those in other C4 grasses, including sorghum. This suggests that Setaria is a suitable model plant for cell wall polysaccharide biology in C4 grasses.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0624-0) contains supplementary material, which is available to authorized users.

The cellulose synthase superfamily in fully sequenced plants and algae

BackgroundThe cellulose synthase superfamily has been classified into nine cellulose synthase-like (Csl) families and one cellulose synthase (CesA) family. The Csl families have been proposed to be involved in the synthesis of the backbones of hemicelluloses of plant cell walls. With 17 plant and algal genomes fully sequenced, we sought to conduct a genome-wide and systematic investigation of this superfamily through in-depth phylogenetic analyses.ResultsA single-copy gene is found in the six chlorophyte green algae, which is most closely related to the CslA and CslC families that are present in the seven land plants investigated in our analyses. Six proteins from poplar, grape and sorghum form a distinct family (CslJ), providing further support for the conclusions from two recent studies. CslB/E/G/H/J families have evolved significantly more rapidly than their widely distributed relatives, and tend to have intragenomic duplications, in particular in the grape genome.ConclusionOur data suggest that the CslA and CslC families originated through an ancient gene duplication event in land plants. We speculate that the single-copy Csl gene in green algae may encode a mannan synthase. We confirm that the rest of the Csl families have a different evolutionary origin than CslA and CslC, and have proposed a model for the divergence order among them. Our study provides new insights about the evolution of this important gene family in plants.

Regulation of the cellulose synthase-like gene family by light in the maize mesocotyl

The cellulose synthase-like (ZmCSL) gene family of maize was annotated and its expression studied in the maize mesocotyl. A total of 28 full-length CSL genes and another 13 partial sequences were annotated; four are predicted to be pseudogenes. Maize has all of the CSL subfamilies that are present in rice, but the CSLC subfamily is expanded from 6 in rice to 12 in maize, and the CSLH subfamily might be reduced from 3 to 1. Unlike rice, maize has a gene in the CSLG subfamily, based on its sequence similarity to two genes annotated as CSLG in poplar. Light regulation of glycan synthase enzyme activities and CSL gene expression were analyzed in the mesocotyl. A Golgi-localized glucan synthase activity is reduced by ~50% 12 h after exposure to light. beta-1,4-Mannan synthase activity is reduced even more strongly (>85%), whereas beta-1,4-xylan synthase, callose synthase, and latent IDPase activity respond only slightly, if at all, to light. At least 17 of the CSL genes (42%) are expressed in the mesocotyl, of which four are up-regulated at least twofold, seven are down-regulated at least twofold, and six are not affected by light. The results contribute to our understanding of the structure of the CSL gene family in an important food and biofuel plant, show that a large percentage of the CSL genes are expressed in the specialized tissues of the mesocotyl, and demonstrate that members of the CSL gene family are differentially subject to photobiological regulation.

Identification of transcripts associated with cell wall metabolism and development in the stem of sugarcane by Affymetrix GeneChip Sugarcane Genome Array expression profiling

Sugarcane is an important crop in tropical regions of the world, producing a very large biomass and accumulating large amounts of sucrose in the stem. In this study, we present the first report of transcript profiling using the GeneChip Sugarcane Genome Array. We have identified transcripts that are differentially expressed in the sugarcane stem during development by expression profiling using the array and total RNA derived from three disparate stem tissues (meristem, internodes 1-3, 8, and 20) from four replicates of the sugarcane variety Q117 grown in the field. We have identified 119 transcripts that were highly differentially expressed with development and have characterised members of the cellulose synthase (CesA) and cellulose synthase-like (Csl) gene families, which displayed coordinated expression during stem development. In addition, we determined that many other transcripts involved in cell wall metabolism and lignification were also co-expressed with members of the CesA and Csl gene families, offering additional insights into the dynamics of primary and secondary cell wall synthesis in the developing sugarcane stem.

Expression of cellulose synthase-like (Csl) genes in insect cells reveals that CslA family members encode mannan synthases.

Glucuronoarabinoxylan, xyloglucan, and galactomannan are noncellulosic polysaccharides found in plant cell walls. All consist of beta-linked glycan backbones substituted with sugar side chains. Although considerable progress has been made in characterizing the structure of these polysaccharides, little is known about the biosynthetic enzymes that produce them. Cellulose synthase-like (Csl) genes are hypothesized to encode Golgi-localized beta-glycan synthases that polymerize the backbones of noncellulosic polysaccharides. To investigate this hypothesis, we used heterologous expression in Drosophila Schneider 2 (S2) cells to systematically analyze the functions of the gene products of a group of Csl genes from Arabidopsis and rice (Oryza sativa L.), including members from five Csl gene families (CslA, CslC, CslD, CslE, and CslH). Our analyses indicate that several members of the CslA gene family encode beta-mannan synthases. Recombinant CslA proteins produce beta-linked mannan polymers when supplied GDP-mannose. The same proteins can produce beta-linked glucomannan heteropolymers when supplied both GDP-mannose and GDP-glucose. One CslA protein also produced beta-linked glucan polymers when supplied GDP-glucose alone. Heterologous expression studies of additional candidate glycan synthases in insect cells or other systems may help identify other noncellulosic polysaccharide biosynthetic enzymes.

Cellulose synthase (CesA) genes in algae and seedless plants

Microfibril structure is determined largely by the organization of arrays of integral plasma membrane protein particles known as “terminal complexes”, which include cellulose synthase catalytic subunits encoded by CesA genes. Although the CesA genes of plants and bacteria share conserved regions, variations in terminal complex and microfibril structure presumably result from sequence differences. Thus, the CesA domains that influence terminal complex assembly may be revealed by examining the differences between CesA genes from green algae in which terminal complex structure ranges from rosettes (plant-like) to linear (bacteria-like). This report describes a second CesA gene that has been cloned from Mesotaenium caldariorum, a unicellular green alga from the order Zygnematales, which have rosette terminal complexes. Both McCesA1 and McCesA2 are similar to seed plant CesAs in domain structure and intron position. Seed plants have multiple CesAs and CesA-like (Csl) genes, some of which appear to be expressed specifically during cell expansion, secondary cell wall deposition in vascular tissue, or tip growth. Diversification of the CesA and Csl gene families can be explored by comparing these genes in mosses, which lack vascular tissue with secondary cell walls, and early divergent vascular plants such as ferns. Degenerate primers were used to amplify and clone five unique CesA and Csl fragments from genomic DNA isolated from Physcomitrella patens. Probes derived from the cloned fragments were used to isolate several clones from a Physcomitrella genomic library. One Csl fragment was amplified from genomic DNA isolated from the fern Ceratopteris richardii. Phylogenetic analysis supports the presence of CslD genes in both mosses and ferns, but does not support the presence of secondary cell wall specific CesA orthologs in mosses.

845 MOLECULAR BASIS OF GROWTH HORMONE (GH) GENE DELETIONS

A rare type of familial isolated GH deficiency (type 1A) is caused by homozygosity for deletion of the structural gene for GH (GH-N). This gene is normally located on the 5' end of the 40-kb GH gene cluster (5'-GH(N)-CS(L)-CS(A)-GH(V)-CS(B)-3'). Six of eight such deletions we have studied differed in either their size (6.7 versus 7.6-kb) or in their pattern of restriction fragments which contain the non-deleted components of the cluster. Since these differences suggested GH-N deletions had occurred multiple different times we analyzed the DNA sequence of a novel fragment produced by a deletion to determine the mechanism of the deletion. The DNA studied was from a subject with a 7.6-kb deletion in which the BAM HI derived 8.3-kb genomic fragment normally containing the CS-L gene was replaced by a unique 9.3-kb fragment. We inferred this unique fragment resulted from fusion of sequences which normally flank the GH-N gene. The complete DNA sequence of the 9.3-kb fragment was determined and the point of transition from sequences normally 5' to GH-N to those normally 3' was detected. The junction of these flanking sequences occurred within the 29-bp sequence TCAGCAGAAGTGGAATTCAGGAGTGAATC. Perfect repeats of these 29 bases lie 497-bp 5' and 7082-bp 3' to the GH-N cap site. Since the 7.6-kb genornic deletions would result from unequal crossing-over between these two homologous copies these repeats may comprise hotspots for recurrence of such deletions.